MDA-9 and uses thereof

ABSTRACT

Increased expression of MDA-9 is associated with drug resistance of certain cells (e.g., cancer cells). The invention provides methods for identifying drug resistant cells by measuring the expression or activity of MDA-9, methods for identifying modulators of drug resistance, and methods for modulating drug resistance by modulating the expression or activity of MDA-9.

RELATED APPLICATION INFORMATION

[0001] This application claims priority from provisional applicationSer. No. 60/125,759, filed Mar. 23, 1999.

BACKGROUND OF THE INVENTION

[0002] The invention relates to chemotherapy and drug resistance.

[0003] Cancer chemotherapy commonly involves the administration of oneor more cytotoxic or cytostatic drugs to a patient. The goal ofchemotherapy is to eradicate a substantially clonal population (tumor)of transformed cells from the body of the individual, or to suppress orto attenuate growth of the tumor. Tumors may occur in solid or liquidform, the latter comprising a cell suspension in blood or other bodyfluid. A secondary goal of chemotherapy is stabilization (clinicalmanagement) of the afflicted individual's health status. Although thetumor may initially respond to chemotherapy, in many instances theinitial chemotherapeutic treatment regimen becomes less effective orceases to impede tumor growth. The selection pressure induced bychemotherapy promotes the development of phenotypic changes that allowtumor cells to resist the cytotoxic effects of a chemotherapeutic drug.Often, exposure to one drug induces resistance to that drug as well asother drugs to which the cells have not been exposed.

SUMMARY OF THE INVENTION

[0004] The present invention concerns melanoma differentiationassociated factor (MDA-9; Genbank Accession No. AF006636; Lin et al.(1996) Mol. Cell. Differ. 4:317-33), a gene which is upregulated in anumber of doxorubicin resistant cell lines (A2780 cells, U937 cells, andHL60 cells). MDA-9 nucleic acids and polypeptides are useful indiagnostic methods related to identification of drug resistant cells(e.g., cancer cells). MDA-9 nucleic acids and polypeptides are alsouseful in screening methods directed to the identification of compoundsthat can modulated (increase or decrease) the drug resistance of aparticular cell type or multiple cell types.

[0005] The invention includes a method for detecting the presence of aMDA-9 polypeptide in a sample. This method features the steps ofcontacting the sample with a compound which selectively binds to thepolypeptide and then determining whether the compound binds to apolypeptide in the sample. In some cases, the compound which binds tothe polypeptide is an antibody.

[0006] The invention also features methods for detecting the presence ofa MDA-9 nucleic acid molecule in a sample. This method includes thesteps of contacting the sample with a nucleic acid probe or primer whichselectively hybridizes to a MDA-9 nucleic acid molecule (e.g., an mRNAencoding MDA-9); and then determining whether the nucleic acid probe orprimer binds to a nucleic acid molecule in the sample.

[0007] Also within the invention are kits that include a compound whichselectively binds to a MDA-9 polypeptide or nucleic acid andinstructions for use. Such kits can be used to determine whether aparticular cell type or cells within a biological sample, e.g., a sampleof patient cells, are drug resistant.

[0008] The invention features methods for identifying a compound whichbinds to a MDA-9 polypeptide. These methods include the steps ofcontacting a MDA-9 polypeptide with a test compound and then determiningwhether the polypeptide binds to the test compound. In variousembodiments of these methods, the binding of the test compound to theMDA-9 polypeptide is detected using an assay which measures binding ofthe test compound to the polypeptide or using a competition bindingassay.

[0009] The invention also includes a method for modulating the activityof a MDA-9 polypeptide. This method includes the steps of contacting thepolypeptide or a cell expressing the polypeptide with a compound whichbinds to the polypeptide in a sufficient concentration to modulate theactivity of the polypeptide.

[0010] In another aspect, the invention provides a method foridentifying a compound that modulates the activity of a MDA-9polypeptide (e.g., a MDA-9 protein). In general, such methods entailmeasuring a biological activity of the polypeptide in the presence andabsence of a test compound and identifying those compounds which alterthe activity of the polypeptide. One such method includes the steps ofcontacting the polypeptide with a test compound and then determining theeffect of the test compound on the activity of the polypeptide tothereby identify a compound which modulates the activity of thepolypeptide.

[0011] The invention also features methods for identifying a compoundwhich modulates the expression of a MDA-9 nucleic acid or a MDA-9polypeptide by measuring the expression of the nucleic acid orpolypeptide in the presence and absence of a compound.

[0012] Other aspects of the invention are methods and compositionsrelating to drug resistance. A “drug-resistant phenotype” refers to acellular phenotype which is associated with increased survival (comparedto a less drug-resistant cell) after exposure to a particular dose of adrug, e.g., a chemotherapeutic drug, compared to a cell that does nothave this phenotype. A “drug-resistant cell” refers to a cell thatexhibits this phenotype. Drug resistance can be characterized by lowerintracellular concentration of a drug compared to a non-resistant cellor a less resistant cell as well as altered ability of a drug to affectits target compared to a non-resistant cell or a less resistant cell.Drug resistance is described in detail by Hochhauser and Harris ((1991)Brit. Med. Bull. 47:178-96); Simon and Schindler ((1994) Proc. Nat'lAcad Sci USA 91: 3497-504); and Harris and Hochhauser ((1992) ActaOncologica 31:205-213); Scotto et. al. ((1986) Science 232: 751-55).Multi-drug resistance can be associated with, for example, alteredcomposition of plasma membrane phospholipids; increased drug binding andintracellular accumulation; altered expression or activity of plasmamembrane or endomembrane channels, transporters or translocators;altered rates of endocytosis and associated alteration in targeting ofendosomes; altered exocytosis; altered intracellular ionic environments;altered expression or activity of proteins involved in drugdetoxification; and altered expression or activity of proteins involvedin DNA repair or replication.

[0013] Also within the invention is a method of determining whether acell has a drug-resistant phenotype by measuring the expression (oractivity) of MDA-9 in the cell and comparing this expression to that ina control cell. Increased expression (or activity) of MDA-9 in the cellcompared to the control cell indicates that the cell has adrug-resistant phenotype. In one embodiment of this method, MDA-9expression is determined by measuring MDA-9 protein (e.g., measuringMDA-9 protein using an antibody directed against MDA-9). In anotherembodiment, MDA-9 expression is measured by quantifying mRNA encodingMDA-9 or the copy number of the MDA-9 gene. In another embodiment MDA-9activity is measured using any assay which can quantify a biologicalactivity of MDA-9.

[0014] The invention also includes a method for modulating the drugresistance of a cell by modulating MDA-9 expression or activity withinthe cell. Thus in one embodiment, the drug-resistance of a cell isreduced by contacting the cell with a molecule (e.g., an antisensenucleic acid molecule) that reduces the expression of MDA-9 within thecell.

[0015] Another aspect of the present invention is a method of improvingeffectiveness of chemotherapy for a mammal having a disorder associatedwith the presence of drug-resistant neoplastic cells. In this method, achemotherapeutic drug and a molecule that reduces expression of MDA-9can be co-administered to a mammal. Alternatively, the chemotherapeuticdrug can be administered before or after administration of the compoundthat reduces expression of MDA-9.

[0016] The invention also includes a method of identifying a compoundthat modulates the drug resistance of a cell by first contacting thecell with a test compound and then measuring and comparing MDA-9expression in the cell exposed to the compound to MDA-9 expression in acontrol cell not exposed to the compound. The compound is identified asmodulator of drug resistance when the level of MDA-9 expression in thecell exposed to the compound differs from the level of MDA-9 expressionin cells not exposed to the compound. In one embodiment of this method,the cell has a drug-resistant phenotype. In another embodiment, the cellis a mammalian cell. This method may also include an optional step ofmeasuring the drug resistance of the cell in the presence of theidentified modulator of drug resistance. The MDA-9 modulating compoundsthat are identified in the foregoing methods are also included withinthe invention.

[0017] The invention also features a method of treating a mammalsuspected of having a disorder associated with the presence ofdrug-resistant cells. This method includes the steps of determiningwhether a mammal has a disorder associated with the presence ofdrug-resistant cells having increased MDA-9 expression (e.g.,drug-resistant cancer), and administering to the mammal a compound thatsufficiently reduces the expression of MDA-9 so that the drug resistanceof the cells associated with the disorder is modulated (i.e., reduced).

[0018] Another feature of the invention is a method for treating apatient having a neoplastic disorder (e.g., cancer) by administering tothe patient a therapeutically effective amount of a compound thatdecreases the expression of MDA-9.

[0019] In the context of cancer treatment, the expression level of MDA-9may be used to: 1) determine if a cancer can be treated by an agent orcombination of agents; 2) determine if a cancer is responding totreatment with an agent or combination of agents; 3) select anappropriate agent or combination of agents for treating a cancer; 4)monitor the effectiveness of an ongoing treatment; and 5) identify newcancer treatments (either single agent or combination of agents). Inparticular, MDA-9 may be used as a marker (surrogate and/or direct) todetermine appropriate therapy, to monitor clinical therapy and humantrials of a drug being tested for efficacy and in developing new agentsand therapeutic combinations.

[0020] Accordingly, the present invention provides methods fordetermining whether an agent, e.g., a chemotherapeutic agent such asdoxorubicin, will be effective in reducing the growth rate of cancercells comprising the steps of: a) obtaining a sample of cancer cells; b)determining the level of expression in the cancer cells of MDA-9; and c)identifying that an agent will be effective when MDA-9 is not expressedor is expressed at relatively low level. Alternatively, in step (c), anagent can be identified as being relatively ineffective when to use totreat the cancer when MDA-9 is expressed or is expressed at relativelyhigh level.

[0021] As used herein, an agent is said to reduce the rate of growth ofcancer cells when the agent can reduce at least 50%, preferably at least75%, most preferably at least 95% of the growth of the cancer cells.Such inhibition can further include a reduction in survivability and anincrease in the rate of death of the cancer cells. The amount of agentused for this determination will vary based on the agent selected.Typically, the amount will be a predefined therapeutic amount.

[0022] As used herein, an agent is defined broadly as anything thatcancer cells can be exposed to in a therapeutic protocol. In the contextof the present invention, such agents include, but are not limited to,chemotherapeutic agents, such as anti-metabolic agents, e.g., Ara AC,5-FU and methotrexate, antimitotic agents, e.g., Taxol, vinblastine andvincristine, alkylating agents, e.g., melphanlan, BCNU and nitrogenmustard, Topoisomerase II inhibitors, e.g., VW-26, topotecan andBleomycin, strand-breaking agents, e.g., doxorubicin and DHAD,cross-linking agents, e.g., cisplatin and CBDCA, radiation andultraviolet light. A preferred agents is doxorubicin.

[0023] The agents tested in the present methods can be a single agent ora combination of agents. For example, the present methods can be used todetermine whether a single chemotherapeutic agent, such as methotrexate,can be used to treat a cancer or whether a combination of two or moreagents can be used.

[0024] Cancer cells include, but are not limited to, carcinomas, such assquamous cell carcinoma, basal cell carcinoma, sweat gland carcinoma,sebaceous gland carcinoma, adenocarcinoma, papillary carcinoma,papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma,undifferentiated carcinoma, bronchogenic carcinoma, melanoma, renal cellcarcinoma, hepatoma-liver cell carcinoma, bile duct carcinoma,cholangiocarcinoma, papillary carcinoma, transitional cell carcinoma,choriocarcinoma, semonoma, embryonal carcinoma, mammary carcinomas,gastrointestinal carcinoma, colonic carcinomas, bladder carcinoma,prostate carcinoma, and squamous cell carcinoma of the neck and headregion; sarcomas, such as fibrosarcoma, myxosarcoma, liposarcoma,chondrosarcoma, osteogenic sarcoma, chordosarcoma, angiosarcoma,endotheliosarcoma, lymphangiosarcoma, synoviosarcoma andmesotheliosarcoma; leukemias and lymphomas such as granulocyticleukemia, monocytic leukemia, lymphocytic leukemia, malignant lymphoma,plasmocytoma, reticulum cell sarcoma, or Hodgkins disease; and tumors ofthe nervous system including glioma, meningoma, medulloblastoma,schwannoma or epidymoma.

[0025] The source of the cancer cells used in the methods of theinvention will be based on how the method of the present invention isbeing used. For example, if the method is being used to determinewhether a patient's cancer can be treated with an agent, or acombination of agents, then the preferred source of cancer cells will becancer cells obtained from a cancer biopsy from the patient.Alternatively, cancer cells line of similar type to that being treatedcan be assayed. For example if breast cancer is being treated, then abreast cancer cell line can be used. If the method is being used tomonitor the effectiveness of a therapeutic protocol, then a tissuesample from the patient being treated is the preferred source. If themethod is being used to identify new therapeutic agents or combinations,then any cancer cells, e.g., cells of a cancer cell line, can be used.

[0026] A skilled artisan can readily select and obtain the appropriatecancer cells that are used in the present method. For cancer cell lines,sources such as The National Cancer Institute, for the NCI-60 cells usedin the examples, are preferred. For cancer cells obtained from apatient, standard biopsy methods, such as a needle biopsy, can beemployed.

[0027] In the methods of the present invention, the level or amount ofexpression of MDA-9 is determined. As used herein, the level or amountof expression refers to the absolute level of expression of an mRNAencoded by the gene or the absolute level of expression of the proteinencoded by the gene (i.e., whether or not expression is or is notoccurring in the cancer cells).

[0028] As an alternative to making determinations based on the absoluteexpression level of selected genes, determinations may be based on thenormalized expression levels. Expression levels are normalized bycorrecting the absolute expression level of a sensitivity or resistancegene by comparing its expression to the expression of a gene that is nota sensitivity or resistance gene, e.g., a housekeeping genes that isconstitutively expressed. Suitable genes for normalization includehousekeeping genes such as the actin gene. This normalization allows oneto compare the expression level in one sample, e.g., a patient sample,to another sample, e.g., a non-cancer sample, or between samples fromdifferent sources. Alternatively, the expression level can be providedas a relative expression level. To determine a relative expression levelof a gene, the level of expression of the gene is determined for 10 ormore samples, preferably 50 or more samples, prior to the determinationof the expression level for the sample in question. The mean expressionlevel of each of the gene assayed in the larger number of samples isdetermined and this is used as a baseline expression level for the genein question. The expression level of the gene determined for the testsample (absolute level of expression) is then divided by the meanexpression value obtained for that gene. This provides a relativeexpression level and aids in identifying extreme cases of sensitivity orresistance. Preferably, the samples used will be from similar tumors orfrom non-cancerous cells of the same tissue origin as the tumor inquestion. The choice of the cell source is dependent on the use of therelative expression level data. For example, using tumors of similartypes for obtaining a mean expression score allows for theidentification of extreme cases of sensitivity or resistance. Usingexpression found in normal tissues as a mean expression score aids invalidating whether the gene assayed is tumor specific (versus normalcells).

[0029] Also within the invention is a method for increasing drugresistance in a cell having an undesirably low level of MDA-9 expressionby administering a compound that increases the expression of MDA-9. Suchmethods are useful for the protection of non-neoplastic cells duringchemotherapy.

[0030] The invention features a method for determining whether a testcompound modulates the drug resistance of a cell, the method including:a) determining the level of MDA-9 expression (e.g., MDA-9 encoded by anendogenous or heterologous gene) in a cell in the presence of a testcompound; b) determining the level of MDA-9 expression in the cell inthe absence of the test compound; and c) identifying the compound as amodulator of drug resistance of the cell if the level of expression ofMDA-9 in the cell in the presence of the test compound differs from thelevel of expression of MDA-9 in the cell in the absence of the testcompound.

[0031] The invention also features a method for determining whether atest compound modulates the drug resistance of a cell, the methodincluding: a) incubating MDA-9 protein in the presence of a testcompound; b) determining whether the test compound binds to the MDA-9protein; c) selecting a test compound which binds to the MDA-9 protein;d) administering the test compound selected in step c) to a non-humanmammal having drug resistant cells; e) determining whether the testcompound alters the drug resistance of the cells in the non-humanmammal; and f) identifying the test compound as a modulator of drugresistance of the cell if the compound alters the drug resistance of thecells in step e).

[0032] The invention further features a method for determining whether atest cell has a drug-resistant phenotype, the method including: a)measuring the expression of MDA-9 in the test cell; b) comparing theexpression of MDA-9 measured in step a) to the expression of MDA-9 in acontrol cell not having a drug-resistant phenotype; and c) determiningthat the test cell has a drug resistant phenotype if the expression ofMDA-9 in the test cell is greater than the expression of MDA-9 in thecontrol cell.

[0033] In another aspect the invention features a method of determiningwhether a test cell has a drug-resistant phenotype, the methodincluding: a) measuring the activity of MDA-9 in the test cell; b)comparing the activity of MDA-9 measured in step a) to the activity ofMDA-9 in a control cell not having a drug-resistant phenotype; and c)determining that the test cell has a drug resistant phenotype if theactivity of MDA-9 in the test cell is greater than the activity of MDA-9in the control cell.

[0034] In yet another aspect the invention features a method fordetermining whether a subject has or is at risk of developing a drugresistant tumor, the method including: a) measuring the expression ofMDA-9 mRNA in a biological sample obtained from the subject (using,e.g., a nucleic acid molecule that hybridizes to MDA-9 mRNA); b)comparing the expression of MDA-9 mRNA measured in step a) to theexpression of MDA-9 mRNA in a biological sample obtained from a controlsubject not having a drug resistant tumor; and c) determining that thepatient has or is at risk of developing a drug resistant tumor if theexpression of MDA-9 mRNA in the biological sample obtained from thepatient is higher than the expression of MDA-9 mRNA in the biologicalsample obtained from the control subject.

[0035] In still another aspect the invention features a method fordetermining whether a subject has or is at risk of developing a drugresistant tumor, the method including: a) measuring the activity ofMDA-9 in a biological sample obtained from the subject (using, e.g., anagent that binds to MDA-9 protein); b) comparing the activity of MDA-9measured in step a) to the expression of MDA-9 mRNA in a biologicalsample obtained from a control subject not having a drug resistanttumor; and c) determining that the patient has or is at risk ofdeveloping a drug resistant tumor if the activity of MDA-9 in thebiological sample obtained from the patient is higher than the activityof MDA-9 in the biological sample obtained from the control subject.

[0036] The invention also features a method for monitoring the effect ofan anti-tumor treatment on a patient, the method including: a) measuringthe expression of MDA-9 in a tumor sample obtained from the patient(using, e.g., a nucleic acid molecule that hybridizes to MDA-9 mRNA); b)comparing the expression of MDA-9 measured in step a) to the expressionof MDA-9 in a control sample of cells; and c) determining that theanti-tumor treatment should be discontinued or modified if theexpression of MDA-9 in the tumor sample is higher than the expression ofMDA-9 in the control sample of cells.

[0037] The invention also features a method for monitoring the effect ofan anti-tumor treatment on a patient, the method including: a) measuringthe activity of MDA-9 in a tumor sample obtained from the patient(using, e.g., an agent that binds to MDA-9 protein); b) comparing theactivity of MDA-9 measured in step a) to the activity of MDA-9 in acontrol sample of cells; and c) determining that the anti-tumortreatment should be discontinued or modified if the activity of MDA-9 inthe tumor sample is higher than the activity of MDA-9 in the controlsample of cells.

[0038] The invention further features a method for modulating the drugresistance of a cell by modulating MDA-9 expression within the cell anda method for reducing the drug resistance of cell by contacting the cellwith a molecule which reduces the expression of MDA-9 within the cell.

[0039] The invention also features a method of increasing theeffectiveness of a chemotherapeutic compound in a patient suffering froma disorder associated with the presence of drug-resistant neoplasticcells, the method including: a) administering a chemotherapeuticcompound to the patient; and b) administering a compound with reducesMDA-9 expression to the patient.

[0040] The invention features a method of treating a mammal suspected ofhaving a disorder associated with the presence of drug-resistant cells,the method including administering to the mammal a compound that reducesthe expression of MDA-9 in the drug-resistant cells, the reduction besufficient to reduce the drug resistance of the drug resistant cells anda method for increasing the drug resistance of cell that has anundesirably low level of MDA-9 expression, the method including exposingthe cell to a compound that increases the expression of MDA-9.

[0041] The invention also features a method for treating a drugresistant tumor in a patient, the method comprising administering tosaid subject an amount of a MDA-9 antagonist effective to reduce drugresistance of said tumor in the patient. In another aspect, theinvention features the use of an inhibitor of MDA-9 expression, orpharmaceutically acceptable salt thereof, or a pharmaceuticalcomposition containing either entity, for the manufacture of amedicament for the treatment of a drug resistant tumor in a patient.

[0042] Unless otherwise defined, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are described herein. All publications, patentapplications, patents, and other references mentioned herein areincorporated by reference in their entirety. In the case of conflict,the present specification, including definitions, will control. Inaddition, the materials, methods, and examples are illustrative only andare not intended to be limiting.

[0043] Other features and advantages of the invention will be apparentfrom the detailed description and from the claims. Although materialsand methods similar or equivalent to those described herein can be usedin the practice or testing of the invention, the preferred materials andmethods are described below.

BRIEF DESCRIPTION OF THE DRAWINGS

[0044]FIG. 1 is a depiction of the nucleotide sequence (SEQ ID NO:1) ofa cDNA encoding human MDA-9 GenBank Accession Mumber AF006636). ThiscDNA encoding human MDA-9 includes an opening reading frame (SEQ IDNO:3) which extends from nucleotide 76 to nucleotide 969 of SEQ IDNO: 1. The start codon and the stop codon are underlined.

[0045]FIG. 2 is a depiction of the predicted amino acid sequence ofhuman MDA-9 (SEQ ID NO:3.

DETAILED DESCRIPTION OF THE INVENTION

[0046] The nucleotide sequence of a cDNA encoding a human MDA-9 protein(SEQ ID NO:1) and the predicted amino acid sequence of human MDA-9protein (SEQ ID NO: 2) is shown in FIGS. 1 and 2 respectively. The openreading frame of the MDA-9 cDNA of FIG. 1 extends from nucleotide 76 tonucleotide 969 of SEQ ID NO:1 (SEQ ID NO:3).

[0047] The association between MDA-9 expression and drug resistance wasdiscovered during a search for genes having was identified as a genethat is more highly expressed in a drug resistant cell line than in themore drug sensitive cell line from which the drug resistant cell linewas derived.

[0048] Various aspects of the invention are described in further detailin the following subsections.

[0049] I. Isolated Nucleic Acid Molecules

[0050] Isolated nucleic acid molecules that encode MDA-9 proteins orbiologically active portions thereof, as well as nucleic acid moleculessufficient for use as hybridization probes to identify MDA-9-encodingnucleic acids (e.g., MDA-9 mRNA) and fragments for use as PCR primersfor the amplification or mutation of MDA-9 nucleic acid molecules, areuseful in the methods of the invention. Various methods for thepreparation and use of MDA-9 nucleic acid molecules are described below.

[0051] As used herein, the term “nucleic acid molecule” is intended toinclude DNA molecules (e.g., cDNA or genomic DNA) and RNA molecules(e.g., mRNA) and analogs of the DNA or RNA generated using nucleotideanalogs. The nucleic acid molecule can be single-stranded ordouble-stranded, but preferably is double-stranded DNA.

[0052] An isolated nucleic acid molecule is one which is separated fromother nucleic acid molecules which are present in the natural source ofthe nucleic acid. Preferably, an isolated nucleic acid is free ofsequences (preferably protein encoding sequences) which naturally flankthe nucleic acid (i.e., sequences located at the 5′ and 3′ ends of thenucleic acid) in the genomic DNA of the organism from which the nucleicacid is derived. An isolated MDA-9 nucleic acid molecule can containless than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb ofnucleotide sequences which naturally flank the nucleic acid molecule ingenomic DNA of the cell from which the nucleic acid is derived.Moreover, an isolated nucleic acid molecule, such as a cDNA molecule,can be substantially free of other cellular material, or culture mediumwhen produced by recombinant techniques, or substantially free ofchemical precursors or other chemicals when chemically synthesized. AMDA-9 nucleic acid molecule, e.g., a nucleic acid molecule having thenucleotide sequence of SEQ ID NO:1 or SEQ ID NO:3, or a complementthereof, can be isolated using standard molecular biology techniques andthe sequence information provided herein. MDA-9 nucleic acid moleculescan be isolated using standard hybridization and cloning techniques(e.g., as described in Sambrook et al., eds., Molecular Cloning: ALaboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).

[0053] A MDA-9 nucleic acid can be amplified using cDNA, mRNA or genomicDNA as a template and appropriate oligonucleotide primers according tostandard PCR amplification techniques. The nucleic acid so amplified canbe cloned into an appropriate vector and characterized by DNA sequenceanalysis. Furthermore, oligonucleotides corresponding to MDA-9nucleotide sequences can be prepared by standard synthetic techniques,e.g., using an automated DNA synthesizer.

[0054] Useful MDA-9 nucleic acid molecules can comprise only a portionof a nucleic acid sequence encoding MDA-9, for example, a fragment whichcan be used as a probe or primer for identifying and/or qualifying MDA-9mRNA in a biological sample. A probe or primer can include at leastabout 12, 25, 50, 75, 100, 125, 150, 175, 200, 250, 300, 350 or 400nucleotides and hybridizes, e.g., under stringent conditions, to a MDA-9mRNA, e.g, an mRNA comprising the nucleotide sequence of SEQ ID NO:1 orSEQ ID NO:3.

[0055] Probes based on the human MDA-9 nucleotide sequence can be usedto detect MDA-9 transcripts or genomic sequences. The probe comprises alabel group attached thereto, e.g., a radioisotope, a fluorescentcompound, an enzyme, or an enzyme co-factor. Such probes can be used asa part of a diagnostic test kit for identifying cells or tissue whichmis-express a MDA-9 protein, such as by measuring a level of aMDA-9-encoding nucleic acid in a sample of cells from a subject, e.g.,detecting MDA-9 mRNA levels or determining whether a genomic MDA-9 genehas been mutated, deleted, or amplified.

[0056] A nucleic acid fragment encoding a “biologically active portionof MDA-9” can be prepared by isolating a portion of SEQ ID NO:3 whichencodes a polypeptide having a MDA-9 biological activity, expressing theencoded portion of MDA-9 protein (e.g., by recombinant expression invitro) and assessing the activity of the encoded portion of MDA-9.

[0057] In addition to the probes and primers described above, isolatednucleic acid molecules of at least 50, 100, 200, 300, 325, 350, 375,400, 425, 450, 500, 550, 600, 650, 700, 800, or 900 nucleotides thathybridize under stringent conditions to a nucleic acid moleculecomprising the nucleotide sequence, preferably the coding sequence ofSEQ ID NO:1 or SEQ ID NO:3 are useful in the methods of the invention.

[0058] As used herein, the term “hybridizes under stringent conditions”is intended to describe conditions for hybridization and washing underwhich nucleotide sequences at least 60% (65%, 70%, preferably 75%)identical to each other typically remain hybridized to each other. Suchstringent conditions are known to those skilled in the art and can befound in Current Protocols in Molecular Biology, John Wiley & Sons, NewYork (1989), 6.3.1-6.3.6. A preferred, non-limiting example of stringenthybridization conditions are hybridization in x sodium chloride/sodiumcitrate (SSC) at about 45□C, followed by one or more washes in 0.2 XSSC, 0.1% SDS at 50-65□C.

[0059] Nucleic acid molecules encoding MDA-9 proteins that containchanges in amino acid residues that are not essential for activity canbe used in the methods of the invention. Such MDA-9 proteins differ inamino acid sequence from SEQ ID NO:2 yet retain biological activity. Forexample, the isolated nucleic acid molecule may include a nucleotidesequence encoding a protein that includes an amino acid sequence that isat least about 45% identical, 65%, 75%, 85%, 95%, or 98% identical tothe amino acid sequence of SEQ ID NO:2.

[0060] An isolated nucleic acid molecule encoding a MDA-9 protein havinga sequence which differs from that of SEQ ID NO:2 can be created byintroducing one or more nucleotide substitutions, additions or deletionsinto the nucleotide sequence of SEQ ID NO:3 such that one or more aminoacid substitutions, additions or deletions are introduced into theencoded protein. Mutations can be introduced by standard techniques,such as site-directed mutagenesis and PCR-mediated mutagenesis.Preferably, conservative amino acid substitutions are made at one ormore predicted non-essential amino acid residues. A “conservative aminoacid substitution” is one in which the amino acid residue is replacedwith an amino acid residue having a similar side chain. Families ofamino acid residues having similar side chains have been defined in theart. These families include amino acids with basic side chains (e.g.,lysine, arginine, histidine), acidic side chains (e.g., aspartic acid,glutamic acid), uncharged polar side chains (e.g., glycine, asparagine,glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains(e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine). Thus, a predicted nonessentialamino acid residue in MDA-9 is preferably replaced with another aminoacid residue from the same side chain family. Alternatively, mutationscan be introduced randomly along all or part of a MDA-9 coding sequence,such as by saturation mutagenesis, and the resultant mutants can bescreened for MDA-9 biological activity to identify mutants that retainactivity. Following mutagenesis, the encoded protein can be expressedrecombinantly and the activity of the protein can be determined.

[0061] Antisense molecules, i.e., molecules which are complementary to asense nucleic acid encoding a protein, e.g., complementary to the codingstrand of a double-stranded cDNA molecule or complementary to an mRNAsequence are useful in the methods of the invention, e.g., for reducingexpression of MDA-9 to reduce the drug resistance of a cell. Theantisense nucleic acid can be complementary to an entire MDA-9 codingstrand, or to only a portion thereof, e.g., all or part of the proteincoding region (or open reading frame). An antisense nucleic acidmolecule can be antisense to a noncoding region of the coding strand ofa nucleotide sequence encoding MDA-9. The noncoding regions (“5′ and 3′untranslated regions”) are the 5′ and 3′ sequences which flank thecoding region and are not translated into amino acids.

[0062] An antisense oligonucleotide can be, for example, about 5, 10,15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length. An antisenseMDA-9 nucleic acid can be constructed using chemical synthesis andenzymatic ligation reactions using procedures known in the art. Forexample, an antisense nucleic acid (e.g., an antisense oligonucleotide)can be chemically synthesized using naturally occurring nucleotides orvariously modified nucleotides designed to increase the biologicalstability of the molecules or to increase the physical stability of theduplex formed between the antisense and sense nucleic acids, e.g.,phosphorothioate derivatives and acridine substituted nucleotides can beused. Examples of modified nucleotides which can be used to generate theantisense nucleic acid include 5-fluorouracil, 5-bromouracil,5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine,5-(carboxyhydroxylmethyl) uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can beproduced biologically using an expression vector into which a nucleicacid has been subcloned in an antisense orientation (i.e., RNAtranscribed from the inserted nucleic acid will be of an antisenseorientation to a target nucleic acid of interest, described further inthe following subsection).

[0063] An antisense nucleic acid molecule is typically administered to asubject or generated in situ such that they hybridize with or bind tocellular mRNA and/or genomic DNA encoding a MDA-9 protein to therebyinhibit expression of the protein, e.g., by inhibiting transcriptionand/or translation. The hybridization can be by conventional nucleotidecomplementarity to form a stable duplex, or, for example, in the case ofan antisense nucleic acid molecule which binds to DNA duplexes, throughspecific interactions in the major groove of the double helix. Anexample of a route of administration of antisense nucleic acid moleculesof the invention include direct injection at a tissue site.Alternatively, antisense nucleic acid molecules can be modified totarget selected cells and then administered systemically. For example,for systemic administration, antisense molecules can be modified suchthat they specifically bind to receptors or antigens expressed on aselected cell surface, e.g., by linking the antisense nucleic acidmolecules to peptides or antibodies which bind to cell surface receptorsor antigens. The antisense nucleic acid molecules can also be deliveredto cells using the vectors described herein. To achieve sufficientintracellular concentrations of the antisense molecules, vectorconstructs in which the antisense nucleic acid molecule is placed underthe control of a strong pol II or pol III promoter are preferred.

[0064] An antisense nucleic acid molecule can be an α-anomeric nucleicacid molecule. An α-anomeric nucleic acid molecule forms specificdouble-stranded hybrids with complementary RNA in which, contrary to theusual β-units, the strands run parallel to each other (Gaultier et al.(1987) Nucleic Acids. Res. 15:6625-6641). The antisense nucleic acidmolecule can also comprise a 2′-o-methylribonucleotide (Inoue et al.(1987) Nucleic Acids Res. 15:6131-6148) or a chimeric RNA-DNA analogue(Inoue et al. (1987) FEBS Lett. 215:327-330).

[0065] Ribozymes, which are catalytic RNA molecules with ribonucleaseactivity which are capable of cleaving a single-stranded nucleic acid,such as an mRNA, to which they have a complementary region can be usedin the methods of the invention. Thus, ribozymes (e.g., hammerheadribozymes (described in Haselhoff and Gerlach (1988) Nature334:585-591)) can be used to catalytically cleave MDA-9 mRNA transcriptsto thereby inhibit translation of MDA-9 mRNA. A ribozyme havingspecificity for a MDA-9-encoding nucleic acid can be designed based uponthe nucleotide sequence of a MDA-9 cDNA disclosed herein (e.g., SEQ IDNO:1, SEQ ID NO:3). For example, a derivative of a Tetrahymena L-19 IVSRNA can be constructed in which the nucleotide sequence of the activesite is complementary to the nucleotide sequence to be cleaved in aMDA-9-encoding mRNA. See, e.g., Cech et al. U.S. Pat. No. 4,987,071; andCech et al. U.S. Pat. No. 5,116,742. Alternatively, MDA-9 mRNA can beused to select a catalytic RNA having a specific ribonuclease activityfrom a pool of RNA molecules. See, e.g., Bartel and Szostak (1993)Science 261:1411-1418.

[0066] Other useful nucleic acid molecules are those which form triplehelical structures. For example, MDA-9 gene expression can be inhibitedby targeting nucleotide sequences complementary to the regulatory regionof the MDA-9 (e.g., the MDA-9 promoter and/or enhancers) to form triplehelical structures that prevent transcription of the MDA-9 gene intarget cells. See generally, Helene (1991) Anticancer Drug Des.6(6):569-84; Helene (1992) Ann. N.Y. Acad. Sci. 660:27-36; and Maher(1992) Bioassays 14(12):807-15.

[0067] Nucleic acid molecules useful in the methods of the invention canbe modified at the base moiety, sugar moiety or phosphate backbone toimprove, e.g., the stability, hybridization, or solubility of themolecule. For example, the deoxyribose phosphate backbone of the nucleicacids can be modified to generate peptide nucleic acids (see Hyrup etal. (1996) Bioorganic & Medicinal Chemistry 4(1): 5-23). As used herein,the terms “peptide nucleic acids” or “PNAs” refer to nucleic acidmimics, e.g., DNA mimics, in which the deoxyribose phosphate backbone isreplaced by a pseudopeptide backbone and only the four naturalnucleobases are retained. The neutral backbone of PNAs has been shown toallow for specific hybridization to DNA and RNA under conditions of lowionic strength. The synthesis of PNA oligomers can be performed usingstandard solid phase peptide synthesis protocols as described in Hyrupet al. (1996) supra; Perry-O'Keefe et al. (1996) Proc. Natl. Acad. Sci.USA 93: 14670-675.

[0068] PNAs of MDA-9 can be used for therapeutic and diagnosticapplications. For example, PNAs can be used as antisense or antigeneagents for sequence-specific modulation of gene expression by, e.g.,inducing transcription or translation arrest or inhibiting replication.PNAs of MDA-9 can also be used, e.g., in the analysis of single basepair mutations in a gene by, e.g., PNA directed PCR clamping; asartificial restriction enzymes when used in combination with otherenzymes, e.g., S1 nucleases (Hyrup (1996) supra; or as probes or primersfor DNA sequence analysis and hybridization (Hyrup (1996) supra;Perry-O'Keefe et al. (1996) Proc. Natl. Acad. Sci. USA 93: 14670-675).

[0069] PNAs of MDA-9 can be modified, e.g., to enhance their stabilityor cellular uptake, by attaching lipophilic or other helper groups toPNA, by the formation of PNA-DNA chimeras, or by the use of liposomes orother techniques of drug delivery known in the art. For example, PNA-DNAchimeras of MDA-9 can be generated which may combine the advantageousproperties of PNA and DNA. Such chimeras allow DNA recognition enzymes,e.g., RNAse H and DNA polymerases, to interact with the DNA portionwhile the PNA portion would provide high binding affinity andspecificity. PNA-DNA chimeras can be linked using linkers of appropriatelengths selected in terms of base stacking, number of bonds between thenucleobases, and orientation (Hyrup (1996) supra). The synthesis ofPNA-DNA chimeras can be performed as described in Hyrup (1996) supra andFinn et al. (1996) Nucleic Acids Research 24(17):3357-63. For example, aDNA chain can be synthesized on a solid support using standardphosphoramidite coupling chemistry and modified nucleoside analogs,e.g., 5′-(4-methoxytrityl)amino-5′-deoxy-thymidine phosphoramidite, canbe used as a linker between the PNA and the 5′ end of DNA (Mag et al.(1989) Nucleic Acid Res. 17:5973-88). PNA monomers are then coupled in astepwise manner to produce a chimeric molecule with a 5′ PNA segment anda 3′ DNA segment (Finn et al. (1996) Nucleic Acids Research24(17):3357-63). Alternatively, chimeric molecules can be synthesizedwith a 5′ DNA segment and a 3′ PNA segment (Peterser et al. (1975)Bioorganic Med. Chem. Lett. 5:1119-11124).

[0070] Useful oligonucleotide may include other appended groups such aspeptides (e.g., for targeting host cell receptors in vivo), or agentsfacilitating transport across the cell membrane (see, e.g., Letsinger etal. (1989) Proc. Natl. Acad. Sci. USA 86:6553-6556; Lemaitre et al.(1987) Proc. Natl Acad. Sci. USA 84:648-652; PCT Publication No.W088/09810) or the blood-brain barrier (see, e.g., PCT Publication No.W089/10134). In addition, oligonucleotides can be modified withhybridization-triggered cleavage agents (See, e.g., Krol et al. (1988)Bio/Techniques 6:958-976) or intercalating agents (See, e.g., Zon (1988)Pharm. Res. 5:539-549). To this end, the oligonucleotide may beconjugated to another molecule, e.g., a peptide, hybridization triggeredcross-linking agent, transport agent, hybridization-triggered cleavageagent, etc.

[0071] II. Isolated MDA-9 Proteins and Anti-MDA-9 Antibodies

[0072] Isolated MDA-9 proteins, and biologically active portionsthereof, as well as polypeptide fragments suitable for use as immunogensto raise anti-MDA-9 antibodies are useful in the methods of theinvention. Methods for the preparation and use of these molecules aredescribed below. In general, MDA-9 proteins can be isolated from cellsor tissue sources by an appropriate purification scheme using standardprotein purification techniques, produced by recombinant DNA techniques,or synthesized chemically using standard peptide synthesis techniques.

[0073] An “isolated” or “purified” protein or biologically activeportion thereof is substantially free of cellular material or othercontaminating proteins from the cell or tissue source from which theMDA-9 protein is derived, or substantially free from chemical precursorsor other chemicals when chemically synthesized. The language“substantially free of cellular material” includes preparations of MDA-9protein in which the protein is separated from cellular components ofthe cells from which it is isolated or recombinantly produced. Thus,MDA-9 protein that is substantially free of cellular material includespreparations of MDA-9 protein having less than about 30%, 20%, 10%, or5% (by dry weight) of non-MDA-9 protein (also referred to herein as a“contaminating protein”). When the MDA-9 protein or biologically activeportion thereof is recombinantly produced, it is also preferablysubstantially free of culture medium, i.e., culture medium representsless than about 20%, 10%, or 5% of the volume of the proteinpreparation. When MDA-9 protein is produced by chemical synthesis, it ispreferably substantially free of chemical precursors or other chemicals,i.e., it is separated from chemical precursors or other chemicals whichare involved in the synthesis of the protein. Accordingly suchpreparations of MDA-9 protein have less than about 30%, 20%, 10%, 5% (bydry weight) of chemical precursors or non-MDA-9 chemicals.

[0074] Biologically active portions of a MDA-9 protein include peptidescomprising amino acid sequences sufficiently identical to or derivedfrom the amino acid sequence of the MDA-9 protein (e.g., the amino acidsequence shown in SEQ ID NO:2), which include less amino acids than thefull length MDA-9 proteins, and exhibit at least one activity of a MDA-9protein. Typically, biologically active portions comprise a domain ormotif with at least one activity of the MDA-9 protein. A biologicallyactive portion of a MDA-9 protein can be a polypeptide which is, forexample, 10, 25, 50, 100 or more amino acids in length.

[0075] Moreover, other biologically active portions, in which otherregions of the protein are deleted, can be prepared by recombinanttechniques and evaluated for one or more of the functional activities ofa native MDA-9 protein. A preferred MDA-9 protein has the amino acidsequence shown of SEQ ID NO:2. Other useful MDA-9 proteins aresubstantially identical to SEQ ID NO:2 and retain the functionalactivity of the protein of SEQ ID NO:2 yet differ in amino acid sequencedue to natural allelic variation or mutagenesis. Accordingly, a usefulMDA-9 protein is a protein which includes an amino acid sequence atleast about 45%, preferably 55%, 65%, 75%, 85%, 95%, or 99% identical tothe amino acid sequence of SEQ ID NO:2 and retains the functionalactivity of the MDA-9 proteins of SEQ ID NO:2.

[0076] To determine the percent identity of two amino acid sequences orof two nucleic acids, the sequences are aligned for optimal comparisonpurposes (e.g., gaps can be introduced in the sequence of a first aminoacid or nucleic acid sequence for optimal alignment with a second aminoor nucleic acid sequence). The amino acid residues or nucleotides atcorresponding amino acid positions or nucleotide positions are thencompared. When a position in the first sequence is occupied by the sameamino acid residue or nucleotide as the corresponding position in thesecond sequence, then the molecules are identical at that position. Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences (i.e., % identity=# ofidentical positions/total# of positions×100).

[0077] The determination of percent homology between two sequences canbe accomplished using a mathematical algorithm. A preferred,non-limiting example of a mathematical algorithm utilized for thecomparison of two sequences is the algorithm of Karlin and Altschul(1990) Proc. Nat'l Acad. Sci. USA 87:2264-2268, modified as in Karlinand Altschul (1993) Proc. Nat'l Acad. Sci. USA 90:5873-5877. Such analgorithm is incorporated into the NBLAST and XBLAST programs ofAltschul, et al. (1990) J. Mol. Biol. 215:403-410. BLAST nucleotidesearches can be performed with the NBLAST program, score=100,wordlength=12 to obtain nucleotide sequences homologous to MDA-9 nucleicacid molecules of the invention. BLAST protein searches can be performedwith the XBLAST program, score=50, wordlength=3 to obtain amino acidsequences homologous to MDA-9 protein molecules of the invention. Toobtain gapped alignments for comparison purposes, Gapped BLAST can beutilized as described in Altschul et al., (1997) Nucleic Acids Res.25:3389-3402. When utilizing BLAST and Gapped BLAST programs, thedefault parameters of the respective programs (e.g., XBLAST and NBLAST)can be used. See http://www.ncbi.nlm.nih.gov. Another preferred,non-limiting example of a mathematical algorithm utilized for thecomparison of sequences is the algorithm of Myers and Miller, CABIOS(1989). Such an algorithm is incorporated into the ALIGN program(version 2.0) which is part of the GCG sequence alignment softwarepackage. When utilizing the ALIGN program for comparing amino acidsequences, a PAM120 weight residue table, a gap length penalty of 12,and a gap penalty of 4 can be used.

[0078] The percent identity between two sequences can be determinedusing techniques similar to those described above, with or withoutallowing gaps. In calculating percent identity, only exact matches arecounted.

[0079] MDA-9 chimeric or fusion proteins are also useful in the methodsof the invention. As used herein, a MDA-9 “chimeric protein” or “fusionprotein” comprises a MDA-9 polypeptide operatively linked to a non-MDA-9polypeptide. A “MDA-9 polypeptide” refers to a polypeptide having anamino acid sequence corresponding to MDA-9, whereas a “non-MDA-9polypeptide” refers to a polypeptide having an amino acid sequencecorresponding to a protein which is not substantially identical to theMDA-9 protein, e.g., a protein which is different from the MDA-9 proteinand which is derived from the same or a different organism. Within aMDA-9 fusion protein the MDA-9 polypeptide can correspond to all or aportion of a MDA-9 protein, preferably at least one biologically activeportion of a MDA-9 protein. Within the fusion protein, the term“operatively linked” is intended to indicate that the MDA-9 polypeptideand the non-MDA-9 polypeptide are fused in-frame to each other. Thenon-MDA-9 polypeptide can be fused to the N-terminus or C-terminus ofthe MDA-9 polypeptide. One useful fusion protein is a GST-MDA-9 fusionprotein in which the MDA-9 sequences are fused to the C-terminus of theGST sequences. Such fusion proteins can facilitate the purification ofrecombinant MDA-9.

[0080] Another useful MDA-9 fusion protein is an MDA-9-immunoglobulinfusion protein in which all or part of MDA-9 is fused to sequencesderived from a member of the immunoglobulin protein family.MDA-9-immunoglobulin fusion proteins of the invention can be used asimmunogens to produce anti-MDA-9 antibodies in a subject, to purifyMDA-9 ligands and in screening assays to identify molecules whichinhibit the interaction of MDA-9 with a protein or nucleic acid whichbinds MDA-9.

[0081] A MDA-9 chimeric or fusion protein can be produced by standardrecombinant DNA techniques. For example, DNA fragments coding for thedifferent polypeptide sequences are ligated together in-frame inaccordance with conventional techniques, for example by employingblunt-ended or stagger-ended termini for ligation, restriction enzymedigestion to provide for appropriate termini, filling-in of cohesiveends as appropriate, alkaline phosphatase treatment to avoid undesirablejoining, and enzymatic ligation. The fusion gene can be synthesized byconventional techniques including automated DNA synthesizers.Alternatively, PCR amplification of gene fragments can be carried outusing anchor primers which give rise to complementary overhangs betweentwo consecutive gene fragments which can subsequently be annealed andreamplified to generate a chimeric gene sequence (see, e.g., CurrentProtocols in Molecular Biology, Ausubel et al. eds., John Wiley & Sons:1992). Moreover, many expression vectors are commercially available thatalready encode a fusion moiety (e.g., a GST polypeptide). AnMDA-9-encoding nucleic acid can be cloned into such an expression vectorsuch that the fusion moiety is linked in-frame to the MDA-9 protein.

[0082] Variants of MDA-9 protein which function as either MDA-9 agonists(mimetics) or as MDA-9 antagonists are useful in the methods of theinvention. Variants of the MDA-9 protein can be generated bymutagenesis, e.g., discrete point mutation or truncation of the MDA-9protein. An agonist of the MDA-9 protein can retain substantially thesame, or a subset, of the biological activities of the naturallyoccurring form of the MDA-9 protein. An antagonist of the MDA-9 proteincan inhibit one or more of the activities of the naturally-occurringform of the MDA-9 protein by, for example, competitively binding topolynucleotides or proteins involved in MDA-9 function. Thus, specificbiological effects can be elicited by treatment with a variant oflimited function. Treatment of a subject with a variant having a subsetof the biological activities of the naturally-occurring form of theprotein can have fewer side effects in a subject relative to treatmentwith the naturally-occurring form of the MDA-9 proteins.

[0083] Variants of the MDA-9 protein which function as either MDA-9agonists (mimetics) or as MDA-9 antagonists can be identified byscreening combinatorial libraries of mutants, e.g., truncation mutants,of the MDA-9 protein for MDA-9 protein agonist or antagonist activity. Alibrary of MDA-9 variants can be produced by, for example, enzymaticallyligating a mixture of synthetic oligonucleotides into gene sequencessuch that a degenerate set of potential MDA-9 sequences is expressibleas individual polypeptides, or alternatively, as a set of larger fusionproteins (e.g., for phage display) containing the set of MDA-9 sequencestherein. There are a variety of methods which can be used to producelibraries of potential MDA-9 variants from a degenerate oligonucleotidesequence. Chemical synthesis of a degenerate gene sequence can beperformed in an automatic DNA synthesizer, and the synthetic gene thenligated into an appropriate expression vector. Use of a degenerate setof genes allows for the provision, in one mixture, of all of thesequences encoding the desired set of potential MDA-9 sequences. Methodsfor synthesizing degenerate oligonucleotides are known in the art (see,e.g., Narang (1983) Tetrahedron 39:3; Itakura et al. (1984) Annu. Rev.Biochem. 53:323; Itakura et al. (1984) Science 198:1056; Ike et al.(1983) Nucleic Acid Res. 11:477).

[0084] In addition, libraries of fragments of the MDA-9 protein codingsequence can be used to generate a variegated population of MDA-9fragments for screening and subsequent selection of variants of a MDA-9protein. For example, a library of coding sequence fragments can begenerated by treating a double stranded PCR fragment of a MDA-9 codingsequence with a nuclease under conditions wherein nicking occurs onlyabout once per molecule, denaturing the double stranded DNA, renaturingthe DNA to form double stranded DNA which can include sense/antisensepairs from different nicked products, removing single stranded portionsfrom reformed duplexes by treatment with S1 nuclease, and ligating theresulting fragment library into an expression vector. By this method, anexpression library can be derived which encodes N-terminal and internalfragments of various sizes of the MDA-9 protein.

[0085] Several techniques are known in the art for screening geneproducts of combinatorial libraries made by point mutations ortruncation, and for screening cDNA libraries for gene products having aselected property. Such techniques are adaptable for rapid screening ofthe gene libraries generated by the combinatorial mutagenesis of MDA-9proteins. The most widely used techniques, which are amenable to highthrough-put analysis, for screening large gene libraries typicallyinclude cloning the gene library into replicable expression vectors,transforming appropriate cells with the resulting library of vectors,and expressing the combinatorial genes under conditions in whichdetection of a desired activity facilitates isolation of the vectorencoding the gene whose product was detected. Recursive ensemblemutagenesis (REM), a technique which enhances the frequency offunctional mutants in the libraries, can be used in combination with thescreening assays to identify MDA-9 variants (Arkin and Yourvan (1992)Proc. Natl. Acad. Sci. USA 89:7811-7815; Delgrave et al. (1993) ProteinEngineering 6(3):327-331).

[0086] An isolated MDA-9 protein, or a portion or fragment thereof, canbe used as an immunogen to generate antibodies that bind MDA-9 usingstandard techniques for polyclonal and monoclonal antibody preparation.The full-length MDA-9 protein can be used or, alternatively, theinvention provides antigenic peptide fragments of MDA-9 for use asimmunogens. The antigenic peptide of MDA-9 comprises at least 8(preferably 10, 15, 20, or 30) amino acid residues of the amino acidsequence shown in SEQ ID NO:2 and encompasses an epitope of MDA-9 suchthat an antibody raised against the peptide forms a specific immunecomplex with MDA-9.

[0087] Preferred epitopes encompassed by the antigenic peptide areregions of MDA-9 that are located on the surface of the protein, e.g.,hydrophilic regions.

[0088] A MDA-9 immunogen typically is used to prepare antibodies byimmunizing a suitable subject, (e.g., rabbit, goat, mouse or othermammal) with the immunogen. An appropriate immunogenic preparation cancontain, for example, recombinantly expressed MDA-9 protein or achemically synthesized MDA-9 polypeptide. The preparation can furtherinclude an adjuvant, such as Freund's complete or incomplete adjuvant,or similar immunostimulatory agent. Immunization of a suitable subjectwith an immunogenic MDA-9 preparation induces a polyclonal anti-MDA-9antibody response.

[0089] Anti-MDA-9 antibodies are useful in the methods of the invention.The term antibody refers to immunoglobulin molecules and immunologicallyactive portions of immunoglobulin molecules, i.e., molecules thatcontain an antigen binding site which specifically binds an antigen,such as MDA-9. A molecule which specifically binds to MDA-9 is amolecule which binds MDA-9, but does not substantially bind othermolecules in a sample, e.g., a biological sample, which naturallycontains MDA-9. Examples of immunologically active portions ofimmunoglobulin molecules include F(ab) and F(ab′)₂ fragments which canbe generated by treating the antibody with an enzyme such as pepsin. Theterm monoclonal antibody or monoclonal antibody composition refers to apopulation of antibody molecules that contain only one species of anantigen binding site capable of immunoreacting with a particular epitopeof MDA-9. A monoclonal antibody composition thus typically displays asingle binding affinity for a particular MDA-9 protein with which itimmunoreacts.

[0090] Polyclonal anti-MDA-9 antibodies can be prepared as describedabove by immunizing a suitable subject with a MDA-9 immunogen. Theanti-MDA-9 antibody titer in the immunized subject can be monitored overtime by standard techniques, such as with an enzyme linked immunosorbentassay (ELISA) using immobilized MDA-9. If desired, the antibodymolecules directed against MDA-9 can be isolated from the mammal (e.g.,from the blood) and further purified by well-known techniques, such asprotein A chromatography to obtain the IgG fraction. At an appropriatetime after immunization, e.g., when the anti-MDA-9 antibody titers arehighest, antibody-producing cells can be obtained from the subject andused to prepare monoclonal antibodies by standard techniques, such asthe hybridoma technique originally described by Kohler and Milstein(1975) Nature 256:495-497, the human B cell hybridoma technique (Kozboret al. (1983) Immunol Today 4:72), the EBV-hybridoma technique (Cole etal. (1985), Monoclonal Antibodies and Cancer Therapy, Alan R. Liss,Inc., pp. 77-96) or trioma techniques. The technology for producingvarious antibodies monoclonal antibody hybridomas is well known (seegenerally Current Protocols in Immunology (1994) Coligan et al. (eds.)John Wiley & Sons, Inc., New York, N.Y.). Briefly, an immortal cell line(typically a myeloma) is fused to lymphocytes (typically splenocytes)from a mammal immunized with a MDA-9 immunogen as described above, andthe culture supernatants of the resulting hybridoma cells are screenedto identify a hybridoma producing a monoclonal antibody that bindsMDA-9.

[0091] Any of the many well known protocols used for fusing lymphocytesand immortalized cell lines can be applied for the purpose of generatingan anti-MDA-9 monoclonal antibody (see, e.g., Current Protocols inImmunology, supra; Galfre et al. (1977) Nature 266:55052; R. H. Kenneth,in Monoclonal Antibodies: A New Dimension In Biological Analyses, PlenumPublishing Corp., New York, N.Y. (1980); and Lerner (1981) Yale J. Biol.Med., 54:387-402. Moreover, the ordinarily skilled worker willappreciate that there are many variations of such methods which alsowould be useful. Typically, the immortal cell line (e.g., a myeloma cellline) is derived from the same mammalian species as the lymphocytes. Forexample, murine hybridomas can be made by fusing lymphocytes from amouse immunized with an immunogenic preparation of the present inventionwith an immortalized mouse cell line, e.g., a myeloma cell line that issensitive to culture medium containing hypoxanthine, aminopterin andthymidine (“HAT medium”). Any of a number of myeloma cell lines can beused as a fusion partner according to standard techniques, e.g., theP3-NS1/1-Ag4-1, P3-x63-Ag8.653 or Sp2/O-Ag14 myeloma lines. Thesemyeloma lines are available from ATCC. Typically, HAT-sensitive mousemyeloma cells are fused to mouse splenocytes using polyethylene glycol(“PEG”). Hybridoma cells resulting from the fusion are then selectedusing HAT medium, which kills unfused and unproductively fused myelomacells (unfused splenocytes die after several days because they are nottransformed). Hybridoma cells producing a monoclonal antibody of theinvention are detected by screening the hybridoma culture supernatantsfor antibodies that bind MDA-9, e.g., using a standard ELISA assay.

[0092] Alternative to preparing monoclonal antibody-secretinghybridomas, a monoclonal anti-MDA-9 antibody can be identified andisolated by screening a recombinant combinatorial immunoglobulin library(e.g., an antibody phage display library) with MDA-9 to thereby isolateimmunoglobulin library members that bind MDA-9. Kits for generating andscreening phage display libraries are commercially available (e.g., thePharmacia Recombinant Phage Antibody System, Catalog No. 27-9400-01; andthe Stratagene SurfZAP™ Phage Display Kit, Catalog No. 240612).Additionally, examples of methods and reagents particularly amenable foruse in generating and screening antibody display library can be foundin, for example, U.S. Pat. No. 5,223,409; PCT Publication No. WO92/18619; PCT Publication No. WO 91/17271; PCT Publication WO 92/20791;PCT Publication No. WO 92/15679; PCT Publication WO 93/01288; PCTPublication No. WO 92/01047; PCT Publication No. WO 92/09690; PCTPublication No. WO 90/02809; Fuchs et al. (1991) Bio/Technology9:1370-1372; Hay et al. (1992) Hum. Antibod. Hybridomas 3:81-85; Huse etal. (1989) Science 246:1275-1281; Griffiths et al. (1993) EMBO J.12:725-734.

[0093] Additionally, recombinant anti-MDA-9 antibodies, such as chimericand humanized monoclonal antibodies, comprising both human and non-humanportions, which can be made using standard recombinant DNA techniques,are within the scope of the invention. Such chimeric and humanizedmonoclonal antibodies can be produced by recombinant DNA techniquesknown in the art, for example using methods described in PCT PublicationNo. WO 87/02671; European Patent Application 184,187; European PatentApplication 171,496; European Patent Application 173,494; PCTPublication No. WO 86/01533; U.S. Pat. No. 4,816,567; European PatentApplication 125,023; Better et al. (1988) Science 240:1041-1043; Liu etal. (1987) Proc. Natl. Acad. Sci. USA 84:3439-3443; Liu et al. (1987) J.Immunol. 139:3521-3526; Sun et al. (1987) Proc. Natl. Acad. Sci. USA84:214-218; Nishimura et al. (1987) Canc. Res. 47:999-1005; Wood et al.(1985) Nature 314:446-449; and Shaw et al. (1988) J. Natl. Cancer Inst.80:1553-1559); Morrison, (1985) Science 229:1202-1207; Oi et al. (1986)Bio/Techniques 4:214; U.S. Pat. No. 5,225,539; Jones et al. (1986)Nature 321:552-525; Verhoeyan et al. (1988) Science 239:1534; andBeidler et al. (1988) J. Immunol. 141:4053-4060.

[0094] An anti-MDA-9 antibody (e.g., monoclonal antibody) can be used toisolate MDA-9 by standard techniques, such as affinity chromatography orimmunoprecipitation. An anti-MDA-9 antibody can facilitate thepurification of natural MDA-9 from cells and of recombinantly producedMDA-9 expressed in host cells. Moreover, an anti-MDA-9 antibody can beused to detect MDA-9 protein (e.g., in a cellular lysate or cellsupernatant) in order to evaluate the abundance and pattern ofexpression of the MDA-9 protein. Anti-MDA-9 antibodies can be useddiagnostically to monitor protein levels in tissue as part of a clinicaltesting procedure, e.g., to, for example, determine the efficacy of agiven treatment regimen. Detection can be facilitated by coupling theantibody to a detectable substance. Examples of detectable substancesinclude various enzymes, prosthetic groups, fluorescent materials,luminescent materials, bioluminescent materials, and radioactivematerials. Examples of suitable enzymes include horseradish peroxidase,alkaline phosphatase, b-galactosidase, or acetylcholinesterase; examplesof suitable prosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerytirin; anexample of a luminescent material includes luminol; examples ofbioluminescent materials include luciferase, luciferin, and aequorin,and examples of suitable radioactive material include ¹²⁵I, ¹³¹I, ³⁵S or³H.

[0095] III. Recombinant Expression Vectors and Host Cells

[0096] Vectors, preferably expression vectors, containing a nucleic acidencoding MDA-9 (or a portion thereof) are useful in the methods of theinvention. A vector is a nucleic acid molecule capable of transportinganother nucleic acid to which it has been linked. One type of vector isa “plasmid”, which refers to a circular double stranded DNA loop intowhich additional DNA segments can be ligated. Another type of vector isa viral vector, wherein additional DNA segments can be ligated into theviral genome. Certain vectors are capable of autonomous replication in ahost cell into which they are introduced (e.g., bacterial vectors havinga bacterial origin of replication and episomal mammalian vectors). Othervectors (e.g., non-episomal mammalian vectors) are integrated into thegenome of a host cell upon introduction into the host cell, and therebyare replicated along with the host genome. Moreover, certain vectors,expression vectors, are capable of directing the expression of genes towhich they are operatively linked. In general, expression vectors ofutility in recombinant DNA techniques are often in the form of plasmids(vectors, e.g., viral vectors, replication defective retroviruses,adenoviruses and adeno-associated viruses).

[0097] Useful recombinant expression vectors comprise a MDA-9 nucleicacid in a form suitable for expression of the nucleic acid in a hostcell, which means that the recombinant expression vectors include one ormore regulatory sequences, selected on the basis of the host cells to beused for expression, which is operatively linked to the nucleic acidsequence to be expressed. Within a recombinant expression vector,“operably linked” is intended to mean that the nucleotide sequence ofinterest is linked to the regulatory sequence(s) in a manner whichallows for expression of the nucleotide sequence (e.g., in an in vitrotranscription/translation system or in a host cell when the vector isintroduced into the host cell). The term “regulatory sequence” isintended to include promoters, enhancers and other expression controlelements (e.g., polyadenylation signals). Such regulatory sequences aredescribed, for example, in Goeddel; Gene Expression Technology: Methodsin Enzymology 185, Academic Press, San Diego, Calif. (1990). Regulatorysequences include those which direct constitutive expression of anucleotide sequence in many types of host cell and those which directexpression of the nucleotide sequence only in certain host cells (e.g.,tissue-specific regulatory sequences). It will be appreciated by thoseskilled in the art that the design of the expression vector can dependon such factors as the choice of the host cell to be transformed, thelevel of expression of protein desired, etc. An expression vector can beintroduced into host cells to thereby produce proteins or peptides,including fusion proteins or peptides, encoded by nucleic acids asdescribed herein (e.g., MDA-9 proteins, mutant forms of MDA-9, fusionproteins, etc.).

[0098] The recombinant expression vectors of the invention can bedesigned for expression of MDA-9 in prokaryotic or eukaryotic cells,e.g., bacterial cells such as E. coli, insect cells (using baculovirusexpression vectors) yeast cells or mammalian cells. Suitable host cellsare discussed further in Goeddel, Gene Expression Technology: Methods inEnzymology 185, Academic Press, San Diego, Calif. (1990). Alternatively,the recombinant expression vector can be transcribed and translated invitro, for example using T7 promoter regulatory sequences and T7polymerase.

[0099] Expression of proteins in prokaryotes is most often carried outin E. coli with vectors containing constitutive or inducible promotersdirecting the expression of either fusion or non-fusion proteins. Fusionvectors add a number of amino acids to a protein encoded therein,usually to the amino terminus of the recombinant protein. Such fusionvectors typically serve three purposes: 1) to increase expression ofrecombinant protein; 2) to increase the solubility of the recombinantprotein; and 3) to aid in the purification of the recombinant protein byacting as a ligand in affinity purification. Often, in fusion expressionvectors, a proteolytic cleavage site is introduced at the junction ofthe fusion moiety and the recombinant protein to enable separation ofthe recombinant protein from the fusion moiety subsequent topurification of the fusion protein. Such enzymes, and their cognaterecognition sequences, include Factor Xa, thrombin and enterokinase.Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc;Smith and Johnson (1988) Gene 67:31-40), pMAL (New England Biolabs,Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) which fuseglutathione S-transferase (GST), maltose E binding protein, or proteinA, respectively, to the target recombinant protein.

[0100] Examples of suitable inducible non-fusion E. coli expressionvectors include pTrc (Amann et al., (1988) Gene 69:301-315) and pET 11d(Studier et al., Gene Expression Technology: Methods in Enzymology 185,Academic Press, San Diego, Calif. (1990) 60-89). Target gene expressionfrom the pTrc vector relies on host RNA polymerase transcription from ahybrid trp-lac fusion promoter. Target gene expression from the pET 11dvector relies on transcription from a T7 gn10-lac fusion promotermediated by a coexpressed viral RNA polymerase (T7 gn1). This viralpolymerase is supplied by host strains BL21(DE3) or HMS174(DE3) from aresident 1 prophage harboring a T7 gn1 gene under the transcriptionalcontrol of the lacUV 5 promoter.

[0101] One strategy to maximize recombinant protein expression in E.coli is to express the protein in a host bacteria with an impairedcapacity to proteolytically cleave the recombinant protein (Gottesman,Gene Expression Technology: Methods in Enzymology 185, Academic Press,San Diego, Calif. (1990) 119-128). Another strategy is to alter thenucleic acid sequence of the nucleic acid to be inserted into anexpression vector so that the individual codons for each amino acid arethose preferentially utilized in E. coli (Wada et al. (1992) NucleicAcids Res. 20:2111-2118). Such alteration of nucleic acid sequences ofthe invention can be carried out by standard DNA synthesis techniques.

[0102] An MDA-9 expression vector is a yeast expression vector. Examplesof vectors for expression in yen be a S. cerivisae include pYepSec1(Baldari et al. (1987) EMBO J. 6:229-234), pMFa (Kurjan and Herskowitz,(1982) Cell 30:933-943), pJRY88 (Schultz et al. (1987) Gene 54:113-123),pYES2 (Invitrogen Corporation, San Diego, Calif.), and picZ (InVitrogenCorp, San Diego, Calif.).

[0103] Alternatively, MDA-9 can be expressed in insect cells usingbaculovirus expression vectors. Baculovirus vectors available forexpression of proteins in cultured insect cells (e.g., Sf 9 cells)include the pAc series (Smith et al. (1983) Mol. Cell Biol. 3:2156-2165)and the pVL series (Lucklow and Summers (1989) Virology 170:31-39).

[0104] An MDA-9 nucleic acid can be expressed in mammalian cells using amammalian expression vector. Examples of mammalian expression vectorsinclude pCDM8 (Seed (1987) Nature 329:840) and pMT2PC (Kaufman et al.(1987) EMBO J. 6:187-195). When used in mammalian cells, the expressionvector's control functions are often provided by viral regulatoryelements. For example, commonly used promoters are derived from polyoma,Adenovirus 2, cytomegalovirus and Simian Virus 40. For other suitableexpression systems for both prokaryotic and eukaryotic cells seechapters 16 and 17 of Sambrook et al. (supra).

[0105] In another embodiment, the recombinant mammalian expressionvector is capable of directing expression of the nucleic acidpreferentially in a particular cell type (e.g., tissue-specificregulatory elements are used to express the nucleic acid).Tissue-specific regulatory elements are known in the art. Non-limitingexamples of suitable tissue-specific promoters include the albuminpromoter (liver-specific; Pinkert et al. (1987) Genes Dev. 1:268-277),lymphoid-specific promoters (Calame and Eaton (1988) Adv. Immunol.43:235-275), in particular promoters of T cell receptors (Winoto andBaltimore (1989) EMBO J. 8:729-733) and immunoglobulins (Banerji et al.(1983) Cell 33:729-740; Queen and Baltimore (1983) Cell 33:741-748),neuron-specific promoters (e.g., the neurofilament promoter; Byrne andRuddle (1989) Proc. Natl. Acad. Sci. USA 86:5473-5477),pancreas-specific promoters (Edlund et al. (1985) Science 230:912-916),and mammary gland-specific promoters (e.g., milk whey promoter; U.S.Pat. No. 4,873,316 and European Application Publication No. 264,166).Developmentally-regulated promoters are also encompassed, for examplethe murine hox promoters (Kessel and Gruss (1990) Science 249:374-379)and the a-fetoprotein promoter (Campes and Tilghman (1989) Genes Dev.3:537-546).

[0106] Also useful in the methods of the invention are recombinantexpression vectors comprising an MDA-9 nucleic acid molecule cloned intothe expression vector in an antisense orientation. That is, the DNAmolecule is operatively linked to a regulatory sequence in a mannerwhich allows for expression (by transcription of the DNA molecule) of anRNA molecule which is antisense to MDA-9 mRNA. Regulatory sequencesoperatively linked to a nucleic acid cloned in the antisense orientationcan be chosen which direct the continuous expression of the antisenseRNA molecule in a variety of cell types, for instance viral promotersand/or enhancers, or regulatory sequences can be chosen which directconstitutive, tissue specific or cell type specific expression ofantisense RNA. The antisense expression vector can be in the form of arecombinant plasmid, phagemid or attenuated virus in which antisensenucleic acids are produced under the control of a high efficiencyregulatory region, the activity of which can be determined by the celltype into which the vector is introduced. For a discussion of theregulation of gene expression using antisense genes See Weintraub etal., Reviews—Trends in Genetics, Vol. 1(1) 1986.

[0107] Host cells into which an MDA-9 expression vector has beenintroduced are useful in certain metods of the invention. The terms“host cell” and “recombinant host cell” are used interchangeably herein.It is understood that such terms refer not only to the particularsubject cell but to the progeny or potential progeny of such a cell.Because certain modifications may occur in succeeding generations due toeither mutation or environmental influences, such progeny may not, infact, be identical to the parent cell, but are still included within thescope of the term as used herein.

[0108] A host cell can be any prokaryotic or eukaryotic cell. Forexample, MDA-9 protein can be expressed in bacterial cells such as E.coli, insect cells, yeast or mammalian cells (such as Chinese hamsterovary cells (CHO) or COS cells). Other suitable host cells are known tothose skilled in the art.

[0109] Vector DNA can be introduced into prokaryotic or eukaryotic cellsvia conventional transformation or transfection techniques. As usedherein, the terms “transformation” and “transfection” are intended torefer to a variety of art-recognized techniques for introducing foreignnucleic acid (e.g., DNA) into a host cell, including calcium phosphateor calcium chloride co-precipitation, DEAE-dextran-mediatedtransfection, lipofection, or electroporation. Suitable methods fortransforming or transfecting host cells can be found in Sambrook, et al.(supra), and other laboratory manuals.

[0110] For stable transfection of mammalian cells, it is known that,depending upon the expression vector and transfection technique used,only a small fraction of cells may integrate the foreign DNA into theirgenome. In order to identify and select these integrants, a gene thatencodes a selectable marker (e.g., resistance to antibiotics) isgenerally introduced into the host cells along with the gene ofinterest. Preferred selectable markers include those which conferresistance to drugs, such as G418, hygromycin and methotrexate. Nucleicacid encoding a selectable marker can be introduced into a host cell onthe same vector as that encoding MDA-9 or can be introduced on aseparate vector. Cells stably transfected with the introduced nucleicacid can be identified by drug selection (e.g., cells that haveincorporated the selectable marker gene will survive, while the othercells die).

[0111] A prokaryotic or eukaryotic host cell in culture can be used toproduce (i.e., express) MDA-9 protein, e.g., by culturing the host cell(into which a recombinant expression vector encoding MDA-9 has beenintroduced) in a suitable medium such that MDA-9 protein is produced.MDA-9 ptoein can then be isolated from the medium or the host cell.

[0112] Host cells which are capable of expressing MDA-9 can also be usedto produce nonhuman transgenic animals. For example, in one embodiment,a host cell of the invention is a fertilized oocyte or an embryonic stemcell into which MDA-9-coding sequences have been introduced. Such hostcells can then be used to create non-human transgenic animals in whichexogenous MDA-9 sequences have been introduced into their genome orhomologous recombinant animals in which endogenous MDA-9 sequences havebeen altered. Such animals are useful for studying the function and/oractivity of MDA-9 and for identifying and/or evaluating modulators ofMDA-9 activity. As used herein, a “transgenic animal” is a non-humananimal, preferably a mammal, more preferably a rodent such as a rat ormouse, in which one or more of the cells of the animal includes atransgene. Other examples of transgenic animals include non-humanprimates, sheep, dogs, cows, goats, chickens, amphibians, etc. Atransgene is exogenous DNA which is integrated into the genome of a cellfrom which a transgenic animal develops and which remains in the genomeof the mature animal, thereby directing the expression of an encodedgene product in one or more cell types or tissues of the transgenicanimal. As used herein, an “homologous recombinant animal” is anon-human animal, preferably a mammal, more preferably a mouse, in whichan endogenous MDA-9 gene has been altered by homologous recombinationbetween the endogenous gene and an exogenous DNA molecule introducedinto a cell of the animal, e.g., an embryonic cell of the animal, priorto development of the animal.

[0113] A transgenic animal can be created by introducing MDA-9-encodingnucleic acid into the male pronuclei of a fertilized oocyte, e.g., bymicroinjection, retroviral infection, and allowing the oocyte to developin a pseudopregnant female foster animal. The MDA-9 cDNA sequence e.g.,that of SEQ ID NO:1 or SEQ ID NO:3 can be introduced as a transgene intothe genome of a non-human animal. Alternatively, a nonhuman homologue ofthe human MDA-9 gene, such as a mouse MDA-9 gene, can be isolated basedon hybridization to the human MDA-9 cDNA and used as a transgene.Intronic sequences and polyadenylation signals can also be included inthe transgene to increase the efficiency of expression of the transgene.A tissue-specific regulatory sequence(s) can be operably linked to theMDA-9 transgene to direct expression of MDA-9 protein to particularcells. Methods for generating transgenic animals via embryo manipulationand microinjection, particularly animals such as mice, have becomeconventional in the art and are described, for example, in U.S. Pat.Nos. 4,736,866 and 4,870,009, U.S. Pat. No. 4,873,191 and in Hogan,Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., 1986). Similar methods are used for productionof other transgenic animals. A transgenic founder animal can beidentified based upon the presence of the MDA-9 transgene in its genomeand/or expression of MDA-9 mRNA in tissues or cells of the animals. Atransgenic founder animal can then be used to breed additional animalscarrying the transgene. Moreover, transgenic animals carrying atransgene encoding MDA-9 can further be bred to other transgenic animalscarrying other transgenes.

[0114] To create an homologous recombinant animal, a vector is preparedwhich contains at least a portion of a MDA-9 gene (e.g., a human or anon-human homolog of the MDA-9 gene, e.g., a murine MDA-9 gene) intowhich a deletion, addition or substitution has been introduced tothereby alter, e.g., functionally disrupt, the MDA-9 gene. In apreferred embodiment, the vector is designed such that, upon homologousrecombination, the endogenous MDA-9 gene is functionally disrupted(i.e., no longer encodes a functional protein; also referred to as a“knock out” vector). Alternatively, the vector can be designed suchthat, upon homologous recombination, the endogenous MDA-9 gene ismutated or otherwise altered but still encodes functional protein (e.g.,the upstream regulatory region can be altered to thereby alter theexpression of the endogenous MDA-9 protein). In the homologousrecombination vector, the altered portion of the MDA-9 gene is flankedat its 5′ and 3′ ends by additional nucleic acid of the MDA-9 gene toallow for homologous recombination to occur between the exogenous MDA-9gene carried by the vector and an endogenous MDA-9 gene in an embryonicstem cell. The additional flanking MDA-9 nucleic acid is of sufficientlength for successful homologous recombination with the endogenous gene.Typically, several kilobases of flanking DNA (both at the 5′ and 3′ends) are included in the vector (see e.g., Thomas and Capecchi (1987)Cell 51:503 for a description of homologous recombination vectors). Thevector is introduced into an embryonic stem cell line (e.g., byelectroporation) and cells in which the introduced MDA-9 gene hashomologously recombined with the endogenous MDA-9 gene are selected (seee.g., Li et al. (1992) Cell 69:915). The selected cells are theninjected into a blastocyst of an animal (e.g., a mouse) to formaggregation chimeras (see, e.g., Bradley in Teratocarcinomas andEmbryonic Stem Cells: A Practical Approach, Robertson, ed. (IRL, Oxford,1987) pp. 113-152). A chimeric embryo can then be implanted into asuitable pseudopregnant female foster animal and the embryo brought toterm. Progeny harboring the homologously recombined DNA in their germcells can be used to breed animals in which all cells of the animalcontain the homologously recombined DNA by germline transmission of thetransgene. Methods for constructing homologous recombination vectors andhomologous recombinant animals are described further in Bradley (1991)Current Opinion in Bio/Technology 2:823-829 and in PCT Publication Nos.WO 90/11354, WO 91/01140, WO 92/0968, and WO 93/04169.

[0115] Transgenic non-human animals can be produced which containselected systems which allow for regulated expression of the transgene.One example of such a system is the cre/loxP recombinase system ofbacteriophage P1. For a description of the cre/loxP recombinase system,see, e.g., Lakso et al. (1992) Proc. Natl. Acad Sci. USA 89:6232-6236.Another example of a recombinase system is the FLP recombinase system ofSaccharomyces cerevisiae (O'Gorman et al. (1991) Science 251:1351-1355.If a cre/loxP recombinase system is used to regulate expression of thetransgene, animals containing transgenes encoding both the Crerecombinase and a selected protein are required. Such animals can beprovided through the construction of “double” transgenic animals, e.g.,by mating two transgenic animals, one containing a transgene encoding aselected protein and the other containing a transgene encoding arecombinase.

[0116] Clones of the non-human transgenic animals described herein canalso be produced according to the methods described in Wilmut et al.(1997) Nature 385:810-813 and PCT Publication Nos. WO 97/07668 and WO97/07669. In brief, a cell, e.g., a somatic cell, from the transgenicanimal can be isolated and induced to exit the growth cycle and enterG_(o) phase. The quiescent cell can then be fused, e.g., through the useof electrical pulses, to an enucleated oocyte from an animal of the samespecies from which the quiescent cell is isolated. The reconstructedoocyte is then cultured such that it develops to morula or blastocyteand then transferred to pseudopregnant female foster animal. Theoffspring borne of this female foster animal will be a clone of theanimal from which the cell, e.g., the somatic cell, is isolated.

[0117] IV. Pharmaceutical Compositions

[0118] MDA-9 proteins, and anti-MDA-9 antibodies, and modulators ofMDA-9 expression or activity (also referred to herein as “activecompounds”) can be incorporated into pharmaceutical compositionssuitable for administration. Such compositions typically comprise thenucleic acid molecule, protein, or antibody and a pharmaceuticallyacceptable carrier. As used herein the language “pharmaceuticallyacceptable carrier” is intended to include any and all solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents, and the like, compatible withpharmaceutical administration. The use of such media and agents forpharmaceutically active substances is well known in the art. Exceptinsofar as any conventional media or agent is incompatible with theactive compound, use thereof in the compositions is contemplated.Supplementary active compounds can also be incorporated into thecompositions.

[0119] A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (topical),transmucosal, and rectal administration. Solutions or suspensions usedfor parenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. pH can beadjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

[0120] Pharmaceutical compositions suitable for injectable use includesterile aqueous solutions (where water soluble) or dispersions andsterile powders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL (BASF; Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyetheylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

[0121] Sterile injectable solutions can be prepared by incorporating theactive compound in the required amount in an appropriate solvent withone or a combination of ingredients enumerated above, as required,followed by filtered sterilization. Generally, dispersions are preparedby incorporating the active compound into a sterile vehicle whichcontains a basic dispersion medium and the required other ingredientsfrom those enumerated above. In the case of sterile powders for thepreparation of sterile injectable solutions, the preferred methods ofpreparation are vacuum drying and freeze-drying which yields a powder ofthe active ingredient plus any additional desired ingredient from apreviously sterile-filtered solution thereof.

[0122] Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring. For administrationby inhalation, the compounds are delivered in the form of an aerosolspray from pressured container or dispenser which contains a suitablepropellant, e.g., a gas such as carbon dioxide, or a nebulizer.

[0123] Systemic administration can also be by transmucosal ortransdermal means. For transmucosal or transdermal administration,penetrants appropriate to the barrier to be permeated are used in theformulation. Such penetrants are generally known in the art, andinclude, for example, for transmucosal administration, detergents, bilesalts, and fusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

[0124] The compounds can also be prepared in the form of suppositories(e.g., with conventional suppository bases such as cocoa butter andother glycerides) or retention enemas for rectal delivery.

[0125] In one embodiment, the active compounds are prepared withcarriers that will protect the compound against rapid elimination fromthe body, such as a controlled release formulation, including implantsand microencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

[0126] It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals.

[0127] MDA-9 nucleic acid molecules of the invention can be insertedinto vectors and used as gene therapy vectors. Gene therapy vectors canbe delivered to a subject by, for example, intravenous injection, localadministration (see U.S. Pat. No. 5,328,470) or by stereotacticinjection (see e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA91:3054-3057). The pharmaceutical preparation of the gene therapy vectorcan include the gene therapy vector in an acceptable diluent, or cancomprise a slow release matrix in which the gene delivery vehicle isimbedded. Alternatively, where the complete gene delivery vector can beproduced intact from recombinant cells, e.g. retroviral vectors, thepharmaceutical preparation can include one or more cells which producethe gene delivery system.

[0128] The pharmaceutical compositions can be included in a container,pack, or dispenser together with instructions for administration.

[0129] V. Uses and Methods of the Invention

[0130] The MDA-9 nucleic acid molecules, proteins, protein homologues,and antibodies described herein can be used in screening assays,predictive medicine (e.g., diagnostic assays, prognostic assays,monitoring clinical trials, and pharmacogenomics), and methods oftreatment (e.g., therapeutic treatment methods and prophylactictreatment methods).

[0131] A. Screening Assays

[0132] The invention provides a method (also referred to herein as a“screening assay”) for identifying modulators, i.e., candidate or testcompounds or agents (e.g., peptides, peptidomimetics, small molecules orother drugs) which bind to MDA-9 proteins or have a stimulatory orinhibitory effect on, for example, MDA-9 expression or MDA-9 activity.Such identified compounds may be useful for the modulation of drugresistance.

[0133] In one embodiment, the invention provides assays for screeningcandidate or test compounds which bind to or modulate the activity of aMDA-9 protein or polypeptide or biologically active portion thereof. Thetest compounds of the present invention can be obtained using any of thenumerous approaches in combinatorial library methods known in the art,including: biological libraries; natural products libraries; spatiallyaddressable parallel solid phase or solution phase libraries; syntheticlibrary methods requiring deconvolution; the ‘one-bead one-compound’library method; and synthetic library methods using affinitychromatography selection. The biological library approach is limited topeptide libraries, while the other approaches are applicable to peptide,non-peptide oligomer or small molecule libraries of compounds (Lam,(1997) Anticancer Drug Des. 12:145).

[0134] Examples of methods for the synthesis of molecular libraries canbe found in the art, for example in: DeWitt et al. (1993) Proc. Natl.Acad. Sci. U.S.A. 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA91:11422; Zucke{grave over ()}rmann et al. (1994). J. Med. Chem.37:2678; Cho et al. (1993) Science 261:1303; Carrell et al. (1994)Angew. Chem. Int. Ed. Engl. 33:2059; Carell et al. (1994) Angew. Chem.Int. Ed. Engl. 33:2061; and Gallop et al. (1994) J. Med. Chem. 37:1233.

[0135] Libraries of compounds may be presented in solution (e.g.,Houghten (1992) Bio/Techniques 13:412-421), or on beads (Lam (19⁹1)Nature 354:82-84), chips (Fodor (1993) Nature 364:555-556), bacteria(U.S. Pat. No. 5,223,409), spores (U.S. Pat. Nos. 5,571,698; 5,403,484;and 5,223,409), plasmids (Cull et al. (1992) Proc. Natl. Acad. Sci. USA89:1865-1869) or on phage (Scott and Smith (1990) Science 249:386-390;Devlin (1990) Science 249:404-406; Cwirla et al. (1990) Proc. Natl.Acad. Sci. 87:6378-6382; and Felici (1991) J. Mol. Biol. 222:301-310).

[0136] In one embodiment, an assay is a cell-based assay in which a cellwhich expresses a MDA-9 protein, or a biologically active portionthereof, is contacted with a test compound and the ability of the testcompound to bind to a MDA-9 protein determined. The cell, for example,can be a yeast cell or a cell of mammalian origin. Determining theability of the test compound to bind to the MDA-9 protein can beaccomplished, for example, by coupling the test compound with aradioisotope or enzymatic label such that binding of the test compoundto the MDA-9 protein or biologically active portion thereof can bedetermined by detecting the labeled compound in a complex. For example,test compounds can be labeled with ¹²⁵I, ³⁵S, ¹⁴C, or ³H, eitherdirectly or indirectly, and the radioisotope detected by direct countingof radioemmission or by scintillation counting. Alternatively, testcompounds can be enzymatically labeled with, for example, horseradishperoxidase, alkaline phosphatase, or luciferase, and the enzymatic labeldetected by determination of conversion of an appropriate substrate toproduct. In a preferred embodiment, the assay comprises contacting acell which expresses a MDA-9 protein, or a biologically active portionthereof, with a known compound which binds MDA-9 to form an assaymixture, contacting the assay mixture with a test compound, anddetermining the ability of the test compound to interact with a MDA-9protein, wherein determining the ability of the test compound tointeract with a MDA-9 protein comprises determining the ability of thetest compound to preferentially bind to MDA-9 or a biologically activeportion thereof as compared to the known compound.

[0137] In another embodiment, an assay is a cell-based assay comprisingcontacting a cell expressing a MDA-9 protein, or a biologically activeportion thereof, with a test compound and determining the ability of thetest compound to modulate (e.g., stimulate or inhibit) the activity ofthe MDA-9 protein or biologically active portion thereof. Determiningthe ability of the test compound to modulate the activity of MDA-9 or abiologically active portion thereof can be accomplished, for example, bydetermining the ability of the MDA-9 protein to bind to or interact witha MDA-9 target molecule. As used herein, a “target molecule” is amolecule with which a MDA-9 protein binds or interacts in nature, forexample, a molecule in the nucleus or cytoplasm of a cell whichexpresses a MDA-9 protein. A MDA-9 target molecule can be a non-MDA-9molecule or a MDA-9 protein or polypeptide. The target, for example, canbe a second intracellular protein which has catalytic activity, aprotein which naturally binds to MDA-9, or a protein which facilitatesthe association of DNA with MDA-9.

[0138] Determining the ability of the MDA-9 protein to bind to orinteract with a MDA-9 target molecule can be accomplished by one of themethods described above for determining direct binding. In a preferredembodiment, determining the ability of the MDA-9 protein to bind to orinteract with a MDA-9 target molecule can be accomplished by determiningthe activity of the target molecule or detecting a cellular response,for example, cell survival or cell proliferation in the presence of achemotherapeutic drug.

[0139] In yet another embodiment, an assay of the present invention is acell-free assay comprising contacting a MDA-9 protein or biologicallyactive portion thereof with a test compound and determining the abilityof the test compound to bind to the MDA-9 protein or biologically activeportion thereof. Binding of the test compound to the MDA-9 protein canbe determined either directly or indirectly as described above. In apreferred embodiment, the assay includes contacting the MDA-9 protein orbiologically active portion thereof with a known compound which bindsMDA-9 to form an assay mixture, contacting the assay mixture with a testcompound, and determining the ability of the test compound to interactwith a MDA-9 protein, wherein determining the ability of the testcompound to interact with a MDA-9 protein comprises determining theability of the test compound to preferentially bind to MDA-9 orbiologically active portion thereof as compared to the known compound.

[0140] In another embodiment, an assay is a cell-free assay comprisingcontacting MDA-9 protein or biologically active portion thereof with atest compound and determining the ability of the test compound tomodulate (e.g., stimulate or inhibit) the activity of the MDA-9 proteinor biologically active portion thereof. Determining the ability of thetest compound to modulate the activity of MDA-9 can be accomplished, forexample, by determining the ability of the MDA-9 protein to bind to aMDA-9 target molecule by one of the methods described above fordetermining direct binding. In an alternative embodiment, determiningthe ability of the test compound to modulate the activity of MDA-9 canbe accomplished by determining the ability of the MDA-9 protein furthermodulate a MDA-9 target molecule. For example, the catalytic/enzymaticactivity of the target molecule on an appropriate substrate can bedetermined as previously described.

[0141] In yet another embodiment, the cell-free assay comprisescontacting the MDA-9 protein or biologically active portion thereof witha known compound which binds MDA-9 to form an assay mixture, contactingthe assay mixture with a test compound, and determining the ability ofthe test compound to interact with a MDA-9 protein, wherein determiningthe ability of the test compound to interact with a MDA-9 proteincomprises determining the ability of the MDA-9 protein to preferentiallybind to or modulate the activity of a MDA-9 target molecule.

[0142] The cell-free assays of the present invention are amenable to useof both native and variant forms (e.g., peptide fragments and fusionproteins) of MDA-9. In the case of cell-free assays comprising ahydrophobic form of MDA-9, it may be desirable to utilize a solubilizingagent such that the hydrophobic form of MDA-9 is maintained in solution.Examples of such solubilizing agents include non-ionic detergents suchas n-octylglucoside, n-dodecylglucoside, n-dodecylmaltoside,octanoyl-N-methylglucamide, decanoyl-N-methylglucamide, Triton® X-100,Triton® X-114, Thesit®), Isotridecypoly(ethylene glycol ether)n,3-[(3-cholamidopropyl)dimethylamminio]-1-propane sulfonate (CHAPS),3-[(3-cholamidopropyl)dimethylamminio]-2-hydroxy-1-propane sulfonate(CHAPSO), or N-dodecyl═N,N-dimethyl-3-ammonio-1-propane sulfonate.

[0143] In more than one embodiment of the above assay methods of thepresent invention, it may be desirable to immobilize either MDA-9 or itstarget molecule to facilitate separation of complexed from uncomplexedforms of one or both of the proteins, as well as to accommodateautomation of the assay. Binding of a test compound to MDA-9, orinteraction of MDA-9 with a target molecule in the presence and absenceof a candidate compound, can be accomplished in any vessel suitable forcontaining the reactants. Examples of such vessels include microtitreplates, test tubes, and micro-centrifuge tubes. In one embodiment, afusion protein can be provided which adds a domain that allows one orboth of the proteins to be bound to a matrix. For example,glutathione-S-transferase/MDA-9 fusion proteins orglutathione-S-transferase/target fusion proteins can be adsorbed ontoglutathione sepharose beads (Sigma Chemical; St. Louis, Mo.) orglutathione derivatized microtitre plates, which are then combined withthe test compound or the test compound and either the non-adsorbedtarget protein or MDA-9 protein, and the mixture incubated underconditions conducive to complex formation (e.g., at physiologicalconditions for salt and pH). Following incubation, the beads ormicrotitre plate wells are washed to remove any unbound components, thematrix immobilized in the case of beads, complex determined eitherdirectly or indirectly, for example, as described above. Alternatively,the complexes can be dissociated from the matrix, and the level of MDA-9binding or activity determined using standard techniques.

[0144] Other techniques for immobilizing proteins on matrices can alsobe used in the screening assays of the invention. For example, eitherMDA-9 or its target molecule can be immobilized utilizing conjugation ofbiotin and streptavidin. Biotinylated MDA-9 or target molecules can beprepared from biotin-NHS (N-hydroxy-succinimide) using techniques wellknown in the art (e.g., biotinylation kit, Pierce Chemicals; Rockford,Ill.), and immobilized in the wells of streptavidin-coated 96 wellplates (Pierce Chemical). Alternatively, antibodies reactive with MDA-9or target molecules but which do not interfere with binding of the MDA-9protein to its target molecule can be derivatized to the wells of theplate, and unbound target or MDA-9 trapped in the wells by antibodyconjugation. Methods for detecting such complexes, in addition to thosedescribed above for the GST-immobilized complexes, includeimmunodetection of complexes using antibodies reactive with the MDA-9 ortarget molecule, as well as enzyme-linked assays which rely on detectingan enzymatic activity associated with the MDA-9 or target molecule.

[0145] In another embodiment, modulators of MDA-9 expression areidentified in a method in which a cell is contacted with a candidatecompound and the expression of MDA-9 (mRNA or protein, or the copynumber of the MDA-9 gene) in the cell is determined. The level ofexpression of MDA-9 in the presence of the candidate compound iscompared to the level of expression of MDA-9 in the absence of thecandidate compound. The candidate compound can then be identified as amodulator of MDA-9 expression based on this comparison. For example,when expression of MDA-9 mRNA or protein is greater (statisticallysignificantly greater) in the presence of the candidate compound than inits absence, the candidate compound is identified as a stimulator ofMDA-9 mRNA or protein expression. Alternatively, when expression ofMDA-9 mRNA or protein is less (statistically significantly less) in thepresence of the candidate compound than in its absence, the candidatecompound is identified as an inhibitor of MDA-9 mRNA or proteinexpression. The level of MDA-9 mRNA or protein expression in the cells,or the number of MDA-9 gene copies per cell can be determined by methodsdescribed herein for detecting MDA-9 genomic DNA, mRNA, or protein.

[0146] MDA-9 proteins can be used as “bait proteins” in a two-hybridassay or three hybrid assay (see, e.g., U.S. Pat. No. 5,283,317; Zervoset al. (1993) Cell 72:223-232; Madura et al. (1993) J. Biol. Chem.268:12046-12054; Bartel et al. (1993) Bio/Techniques 14:920-924;Iwabuchi et al. (1993) Oncogene 8:1693-1696; and WO94/10300), toidentify other proteins, which bind to or interact with MDA-9(“MDA-9-binding proteins” or “MDA-9-bp”) and modulate MDA-9 activity.

[0147] The two-hybrid system is based on the modular nature of mosttranscription factors, which consist of separable DNA-binding andactivation domains. Briefly, the assay utilizes two different DNAconstructs. In one construct, the gene that codes for MDA-9 is fused toa gene encoding the DNA binding domain of a known transcription factor(e.g., GAL-4). In the other construct, a DNA sequence, from a library ofDNA sequences, that encodes an unidentified protein (“prey” or “sample”)is fused to a gene that codes for the activation domain of the knowntranscription factor. If the “bait” and the “prey” proteins are able tointeract, in vivo, forming an MDA-9-dependent complex, the DNA-bindingand activation domains of the transcription factor are brought intoclose proximity. This proximity allows transcription of a reporter gene(e.g., LacZ) which is operably linked to a transcriptional regulatorysite responsive to the transcription factor. Expression of the reportergene can be detected and cell colonies containing the functionaltranscription factor can be isolated and used to obtain the cloned genewhich encodes the protein which interacts with MDA-9.

[0148] This invention further pertains to novel agents identified by theabove-described screening assays and uses thereof for treatments asdescribed herein.

[0149] B. Predictive Medicine

[0150] The present invention also pertains to the field of predictivemedicine in which diagnostic assays, prognostic assays,pharmacogenomics, and monitoring clinical trials are used for prognostic(predictive) purposes to thereby treat an individual prophylactically.Accordingly, one aspect of the present invention relates to diagnosticassays for determining MDA-9 protein and/or nucleic acid expression aswell as MDA-9 activity, in the context of a biological sample (e.g.,blood, serum, cells, tissue) to thereby determine whether an individualis afflicted with a disease or disorder, or is at risk of developing adisorder, associated with aberrant MDA-9 expression or activity (e.g.,altered drug resistance). The invention also provides for prognostic (orpredictive) assays for determining whether an individual is at risk ofdeveloping a disorder associated with MDA-9 protein, nucleic acidexpression or activity (e.g., altered drug resistance). For example,mutations in a MDA-9 gene can be assayed in a biological sample. Suchassays can be used for prognostic or predictive purpose to therebyprophylactically treat an individual prior to the onset of a disordercharacterized by or associated with MDA-9 protein, nucleic acidexpression or activity. For example, because MDA-9 is expressed at ahigher level in drug resistant cells (e.g., the doxorubicin resistantcell lines A2780, U937, and HL60) than non-drug resistant cell lines,higher than normal expression of MDA-9 can be used as an indicator ofdrug resistance.

[0151] Another aspect of the invention provides methods for determiningMDA-9 protein, nucleic acid expression or MDA-9 activity in anindividual to thereby select appropriate therapeutic or prophylacticagents for that individual (referred to herein as “pharmacogenomics”).Pharmacogenomics allows for the selection of agents (e.g., drugs) fortherapeutic or prophylactic treatment of an individual based on thegenotype of the individual (e.g., the genotype of the individualexamined to determine the ability of the individual to respond to aparticular agent.)

[0152] Yet another aspect of the invention pertains to monitoring theinfluence of agents (e.g., drugs or other compounds) on the expressionor activity of MDA-9 in clinical trials.

[0153] These and other agents are described in further detail in thefollowing sections.

[0154] 1. Diagnostic Assays

[0155] The invention provides a method of assessing expression,especially undesirable expression, of a cellular MDA-9 gene. Undesirable(e.g., excessive) expression may indicate the presence, persistence orreappearance of drug-resistant (e.g., doxorubicin-resistant) tumor cellsin an individual's tissue. More generally, aberrant expression mayindicate the occurrence of a deleterious or disease-associated phenotypecontributed to by MDA-9.

[0156] An exemplary method for detecting the presence or absence ofMDA-9 in a biological sample involves obtaining a biological sample(preferably from a body site implicated in a possible diagnosis ofdiseased or malignant tissue) from a test subject and contacting thebiological sample with a compound or an agent capable of detecting MDA-9protein or nucleic acid (e.g., mRNA, genomic DNA) that encodes MDA-9protein such that the presence of MDA-9 is detected in the biologicalsample. The presence and/or relative abundance of MDA-9 indicatesaberrant or undesirable expression of a cellular MDA-9 gene, andcorrelates with the occurrence in situ of cells having a drug-resistantphenotype.

[0157] A preferred agent for detecting MDA-9 mRNA or genomic DNA is alabeled nucleic acid probe capable of hybridizing to MDA-9 mRNA orgenomic DNA. The nucleic acid probe can be, for example, a full-lengthMDA-9 nucleic acid, such as the nucleic acid of SEQ ID NO: 1 or 3, or aportion thereof, such as an oligonucleotide of at least 15, 30, 50, 100,250 or 500 nucleotides in length and sufficient to specificallyhybridize under stringent conditions to MDA-9 mRNA or genomic DNA. Othersuitable probes for use in the diagnostic assays of the invention aredescribed herein.

[0158] A preferred agent for detecting MDA-9 protein is an antibodycapable of binding to MDA-9 protein, preferably an antibody with adetectable label. Antibodies can be polyclonal, or more preferably,monoclonal. An intact antibody, or a fragment thereof (e.g., Fab orF(ab′)₂) can be used. The term “labeled”, with regard to the probe orantibody, is intended to encompass direct labeling of the probe orantibody by coupling (i.e., physically linking) a detectable substanceto the probe or antibody, as well as indirect labeling of the probe orantibody by reactivity with another reagent that is directly labeled.Examples of indirect labeling include detection of a primary antibodyusing a fluorescently labeled secondary antibody and end-labeling of aDNA probe with biotin such that it can be detected with fluorescentlylabeled streptavidin. The term “biological sample” is intended toinclude tissues, cells and biological fluids isolated from a subject, aswell as tissues, cells and fluids present within a subject. That is, thedetection method of the invention can be used to detect MDA-9 mRNA,protein, or genomic DNA in a biological sample in vitro as well as invivo. For example, in vitro techniques for detection of MDA-9 mRNAinclude Northern hybridizations and in situ hybridizations. In vitrotechniques for detection of MDA-9 protein include enzyme linkedimmunosorbent assays (ELISAs), Western blots, immunoprecipitation andimmunofluorescence. In vitro techniques for detection of MDA-9 genomicDNA include Southern hybridizations.

[0159] In one embodiment, the biological sample contains proteinmolecules from the test subject. Alternatively, the biological samplecan contain mRNA molecules from the test subject or genomic DNAmolecules from the test subject. A preferred biological sample is aperipheral blood leukocyte sample isolated by conventional means from asubject.

[0160] In another embodiment, the methods further involve obtaining acontrol biological sample from a control subject, contacting the controlsample with a compound or agent capable of detecting MDA-9 protein,mRNA, or genomic DNA, such that the presence of MDA-9 protein, mRNA orgenomic DNA is detected in the biological sample, and comparing thepresence of MDA-9 protein, mRNA or genomic DNA in the control samplewith the presence of MDA-9 protein, mRNA or genomic DNA in the testsample.

[0161] The invention also encompasses kits for detecting the presence ofMDA-9 in a biological sample (a test sample). Such kits can be used todetermine if a subject is suffering from or is at increased risk ofdeveloping a disorder associated with aberrant expression of MDA-9(e.g., the presence of a drug resistance cancer). For example, the kitcan comprise a labeled compound or agent capable of detecting MDA-9protein or mRNA in a biological sample and means for determining theamount of MDA-9 in the sample (e.g., an anti-MDA-9 antibody or anoligonucleotide probe which binds to DNA encoding MDA-9, e.g., SEQ IDNO:1 or SEQ ID NO:3). Kits may also include instruction for observingthat the tested subject is suffering from or is at risk of developing adisorder associated with aberrant expression of MDA-9 if the amount ofMDA-9 protein or mRNA is above or below a normal level.

[0162] For antibody-based kits, the kit may comprise, for example: (1) afirst antibody (e.g., attached to a solid support) which binds to MDA-9protein; and, optionally, (2) a second, different antibody which bindsto MDA-9 protein or the first antibody and is conjugated to a detectableagent.

[0163] For oligonucleotide-based kits, the kit may comprise, forexample: (1) a oligonucleotide, e.g., a detectably labelledoligonucleotide, which hybridizes to a MDA-9 nucleic acid sequence or(2) a pair of primers useful for amplifying a MDA-9 nucleic acidmolecule;

[0164] The kit may also comprise, e.g., a buffering agent, apreservative, or a protein stabilizing agent. The kit may also comprisecomponents necessary for detecting the detectable agent (e.g., an enzymeor a substrate). The kit may also contain a control sample or a seriesof control samples which can be assayed and compared to the test samplecontained. Each component of the kit is usually enclosed within anindividual container and all of the various containers are within asingle package along with instructions for observing whether the testedsubject is suffering from or is at risk of developing a disorderassociated with aberrant expression of MDA-9.

[0165] 2. Prognostic Assays

[0166] The methods described herein can furthermore be utilized asdiagnostic or prognostic assays to identify subjects having or at riskof developing a disease or disorder associated with aberrant MDA-9expression or activity. For example, the assays described herein, suchas the preceding diagnostic assays or the following assays, can beutilized to identify a subject having or at risk of developing adisorder associated with aberrant MDA-9 protein, nucleic acid expressionor activity (eg., the presence of drug resistant tumor cells).Alternatively, the prognostic assays can be utilized to identify asubject having or at risk for developing such a disease or disorder.Thus, the present invention provides a method in which a test sample isobtained from a subject and MDA-9 protein or nucleic acid (e.g., mRNA,genomic DNA) is detected, wherein the presence or relative quantity ofMDA-9 protein or nucleic acid is diagnostic for a subject having or atrisk of developing a disease or disorder associated with aberrant MDA-9expression or activity. As used herein, a “test sample” refers to abiological sample obtained from a subject of interest. For example, atest sample can be a biological fluid (e.g., serum), cell sample, ortissue.

[0167] Furthermore, the prognostic assays described herein can be usedto determine whether a subject can be administered an agent (e.g., anagonist, antagonist, peptidomimetic, protein, peptide, nucleic acid,small molecule, or other drug candidate) to treat a disease or disorderassociated with aberrant MDA-9 expression or activity. Thus, ifincreased MDA-9 expression is a cause of increased drug resistance, suchmethods can be used to determine whether a subject can be effectivelytreated with a specific agent or class of agents (e.g., agents of a typewhich decrease MDA-9 activity). Thus, the present invention providesmethods for determining whether a subject can be effectively treatedwith an agent for a disorder associated with aberrant MDA-9 expressionor activity in which a test sample is obtained and MDA-9 protein ornucleic acid is detected (e.g., wherein the presence or relativequantity of MDA-9 protein or nucleic acid is diagnostic for a subjectthat can be administered the agent to treat a disorder associated withaberrant MDA-9 expression or activity). In some embodiments, theforegoing methods provide information useful in prognostication, stagingand management of malignancies (tumors) that are characterized byaltered expression of MDA-9 and thus by a drug-resistance phenotype. Theinformation more specifically assists the clinician in designingchemotherapeutic or other treatment regimes to eradicate suchmalignancies from the body of an afflicted subject.

[0168] The methods of the invention can also be used to detect geneticlesions (e.g., mutations or amplifications) in a MDA-9 gene, therebydetermining if a subject with the altered gene is at risk for a disordercharacterized by aberrant cell proliferation and/or differentiation. Forexample, genetic mutations, whether of germline or somatic origin, mayindicate whether the process of developing drug resistance has beeninitiated or is likely to arise in the tested cells. In preferredembodiments, the methods include detecting, in a sample of cells fromthe subject, the presence or absence of a genetic lesion characterizedby at least one of an alteration affecting the integrity of a geneencoding a MDA-9-protein, the mis-expression of the MDA-9 gene, or theamplification of a MDA-9 gene. Preferably the sample of cells isobtained from a body tissue suspected of comprising transformed cells(e.g., cancer cells). Thus, the present method provides informationrelevant to diagnosis of the presence of a tumor.

[0169] Genetic lesions can be detected, for example, by ascertaining theexistence of at least one of 1) a deletion of one or more nucleotidesfrom a MDA-9 gene; 2) an addition of one or more nucleotides to a MDA-9gene; 3) a substitution of one or more nucleotides of a MDA-9 gene, 4) achromosomal rearrangement of a MDA-9 gene; 5) an alteration in the levelof a messenger RNA transcript of a MDA-9 gene, 6) aberrant modificationof a MDA-9 gene, such as of the methylation pattern of the genomic DNA,7) the presence of a non-wild type splicing pattern of a messenger RNAtranscript of a MDA-9 gene, 8) a non-wild type level of a MDA-9-protein,9) allelic loss of a MDA-9 gene, 10) amplification of a MDA-9 gene, and11) inappropriate post-translational modification of a MDA-9-protein. Asdescribed herein, there are a large number of assay techniques known inthe art which can be used for detecting lesions in a MDA-9 gene. Apreferred biological sample is a biopsy sample of tissue suspected ofcomprising transformed cells isolated by conventional means from asubject.

[0170] In certain embodiments, detection of the lesion involves the useof a probe/primer in a polymerase chain reaction (PCR) (see, e.g., U.S.Pat. Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or,alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegranet al. (1988) Science 241:1077-1080; and Nakazawa et al. (1994) Proc.Natl. Acad. Sci. USA 91:360-364), the latter of which can beparticularly useful for detecting point mutations in the MDA-9 gene (seeAbravaya et al. (1995) Nucleic Acids Res. 23:675-682). This method caninclude the steps of collecting a sample of cells from a patient,isolating nucleic acid (e.g., genomic, mRNA or both) from the cells ofthe sample, contacting the nucleic acid sample with one or more primerswhich specifically hybridize to a MDA-9 gene under conditions such thathybridization and amplification of the MDA-9-gene (if present) occurs,and detecting the presence or absence of an amplification product, ordetecting the size of the amplification product and comparing the lengthto a control sample. It is anticipated that PCR and/or LCR may bedesirable to use as a preliminary amplification step in conjunction withany of the techniques used for detecting mutations described herein.

[0171] Alternative amplification methods include: self-sustainedsequence replication (Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA87:1874-1878), transcriptional amplification system (Kwoh, et al. (1989)Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase (Lizardi etal. (1988) Bio/Technology 6:1197), or any other nucleic acidamplification method, followed by the detection of the amplifiedmolecules using techniques well known to those of skill in the art.These detection schemes are especially useful for the detection ofnucleic acid molecules if such molecules are present in very lownumbers.

[0172] In an alternative embodiment, mutations in a MDA-9 gene from asample cell can be identified by alterations in restriction enzymecleavage patterns. For example, sample and control DNA is isolated,amplified (optionally), digested with one or more restrictionendonucleases, and fragment length sizes are determined by gelelectrophoresis and compared. Differences in fragment length sizesbetween sample and control DNA indicates mutations in the sample DNA.Moreover, the use of sequence specific ribozymes (see, e.g., U.S. Pat.No. 5,498,531) can be used to score for the presence of specificmutations by development or loss of a ribozyme cleavage site.

[0173] In other embodiments, genetic mutations in MDA-9 can beidentified by hybridizing a sample and control nucleic acids, e.g., DNAor RNA, to high density arrays containing hundreds or thousands ofoligonucleotides probes (Cronin et al. (1996) Human Mutation 7:244-255;Kozal et al. (1996) Nature Medicine 2:753-759). For example, geneticmutations in MDA-9 can be identified in two-dimensional arrayscontaining light-generated DNA probes as described in Cronin et al.supra. Briefly, a first hybridization array of probes can be used toscan through long stretches of DNA in a sample and control to identifybase changes between the sequences by making linear arrays of sequentialoverlapping probes. This step allows the identification of pointmutations. This step is followed by a second hybridization array thatallows the characterization of specific mutations by using smaller,specialized probe arrays complementary to all variants or mutationsdetected. Each mutation array is composed of parallel probe sets, onecomplementary to the wild-type gene and the other complementary to themutant gene.

[0174] In yet another embodiment, any of a variety of sequencingreactions known in the art can be used to directly sequence the MDA-9gene and detect mutations by comparing the sequence of the sample MDA-9with the corresponding wild-type (control) sequence. Additionally,sequencing of the DNA flanking the MDA-9 can be used to determine if theMDA-9 gene has been amplified. Examples of sequencing reactions includethose based on techniques developed by Maxim and Gilbert ((1977) Proc.Natl. Acad. Sci. USA 74:560) or Sanger ((1977) Proc. Natl. Acad. Sci USA74:5463). It is also contemplated that any of a variety of automatedsequencing procedures can be utilized when performing the diagnosticassays ((1995) Bio/Techniques 19:448), including sequencing by massspectrometry (see, e.g., PCT Publication No. WO 94/16101; Cohen et al.(1996) Adv. Chromatogr. 36:127-162; and Griffin et al. (1993) Appl.Biochem. Biotechnol. 38:147-159).

[0175] Other methods for detecting mutations in the MDA-9 gene includemethods in which protection from cleavage agents is used to detectmismatched bases in RNA/RNA or RNA/DNA heteroduplexes (Myers et al.(1985) Science 230:1242). In general, the art technique of “mismatchcleavage” starts by providing heteroduplexes of formed by hybridizing(labeled) RNA or DNA containing the wild-type MDA-9 sequence withpotentially mutant RNA or DNA obtained from a tissue sample. Thedouble-stranded duplexes are treated with an agent which cleavessingle-stranded regions of the duplex such as which will exist due tobasepair mismatches between the control and sample strands. Forinstance, RNA/DNA duplexes can be treated with RNase and DNA/DNA hybridstreated with S1 nuclease to enzymatically digesting the mismatchedregions. In other embodiments, either DNA/DNA or RNA/DNA duplexes can betreated with hydroxylamine or osmium tetroxide and with piperidine inorder to digest mismatched regions. After digestion of the mismatchedregions, the resulting material is then separated by size on denaturingpolyacrylamide gels to determine the site of mutation. See, e.g., Cottonet al (1988) Proc. Natl Acad Sci USA 85:4397; Saleeba et al (1-992)Methods Enzymol. 217:286-295. In a preferred embodiment, the control DNAor RNA can be labeled for detection.

[0176] In still another embodiment, the mismatch cleavage reactionemploys one or more proteins that recognize mismatched base pairs indouble-stranded DNA (so called “DNA mismatch repair” enzymes) in definedsystems for detecting and mapping point mutations in MDA-9 cDNAsobtained from samples of cells. For example, the mutY enzyme of E. colicleaves A at G/A mismatches and the thymidine DNA glycosylase from HeLacells cleaves T at G/T mismatches (Hsu et al. (1994) Carcinogenesis15:1657-1662). According to an exemplary embodiment, a probe based on aMDA-9 sequence, e.g., a wild-type MDA-9 sequence, is hybridized to acDNA or other DNA product from a test cell(s). The duplex is treatedwith a DNA mismatch repair enzyme, and the cleavage products, if any,can be detected from electrophoresis protocols or the like. See, e.g.,U.S. Pat. No. 5,459,039.

[0177] In other embodiments, alterations in electrophoretic mobilitywill be used to identify mutations in MDA-9 genes. For example, singlestrand conformation polymorphism (SSCP) may be used to detectdifferences in electrophoretic mobility between mutant and wild typenucleic acids (Orita et al. (1989) Proc Natl. Acad. Sci USA: 86:2766,see also Cotton (1993) Mutat. Res. 285:125-144; and Hayashi (1992) GenetAnal Tech Appl 9:73-79). Single-stranded DNA fragments of sample andcontrol MDA-9 nucleic acids will be denatured and allowed to renature.The secondary structure of single-stranded nucleic acids variesaccording to sequence, the resulting alteration in electrophoreticmobility enables the detection of even a single base change. The DNAfragments may be labeled or detected with labeled probes. Thesensitivity of the assay may be enhanced by using RNA (rather than DNA),in which the secondary structure is more sensitive to a change insequence. In a preferred embodiment, the subject method utilizesheteroduplex analysis to separate double stranded heteroduplex moleculeson the basis of changes in electrophoretic mobility (Keen et al. (1991)Trends Genet 7:5).

[0178] In yet another embodiment, the movement of mutant or wild-typefragments in polyacrylamide gels containing a gradient of denaturant isassayed using denaturing gradient gel electrophoresis (DGGE) (Myers etal. (1985) Nature 313:495). When DGGE is used as the method of analysis,DNA will be modified to insure that it does not completely denature, forexample by adding a GC clamp of approximately 40 bp of high-meltingGC-rich DNA by PCR. In a further embodiment, a temperature gradient isused in place of a denaturing gradient to identify differences in themobility of control and sample DNA (Rosenbaum and Reissner (1987)Biophys Chem 265:12753).

[0179] Examples of other techniques for detecting point mutationsinclude, but are not limited to, selective oligonucleotidehybridization, selective amplification, or selective primer extension.For example, oligonucleotide primers may be prepared in which the knownmutation is placed centrally and then hybridized to target DNA underconditions which permit hybridization only if a perfect match is found(Saiki et al. (1986) Nature 324:163); Saiki et al. (1989) Proc. NatlAcad. Sci USA 86:6230). Such allele specific oligonucleotides arehybridized to PCR amplified target DNA or a number of differentmutations when the oligonucleotides are attached to the hybridizingmembrane and hybridized with labeled target DNA.

[0180] Alternatively, allele specific amplification technology whichdepends on selective PCR amplification may be used in conjunction withthe instant invention. Oligonucleotides used as primers for specificamplification may carry the mutation of interest in the center of themolecule (so that amplification depends on differential hybridization)(Gibbs et al. (1989) Nucleic Acids Res. 17:2437-2448) or at the extreme3′ end of one primer where, under appropriate conditions, mismatch canprevent, or reduce polymerase extension (Prossner (1993) Tibtech11:238). In addition, it may be desirable to introduce a novelrestriction site in the region of the mutation to create cleavage-baseddetection (Gasparini et al. (1992) Mol. Cell Probes 6:1). It isanticipated that in certain embodiments amplification may also beperformed using Taq ligase for amplification (Barany (1991) Proc. Natl.Acad. Sci USA 88:189). In such cases, ligation will occur only if thereis a perfect match at the 3′ end of the 5′ sequence making it possibleto detect the presence of a known mutation at a specific site by lookingfor the presence or absence of amplification.

[0181] The methods described herein may be performed, for example, byutilizing pre-packaged diagnostic kits comprising at least one probenucleic acid or antibody reagent described herein, which may beconveniently used, e.g., in clinical settings to diagnose patientsexhibiting symptoms or family history of a disease or illness involvinga MDA-9 gene.

[0182] Furthermore, any cell type or tissue, preferably biopsy samplesof tissue comprising or suspected of comprising transformed cells, inwhich MDA-9 is expressed may be utilized in the prognostic assaysdescribed herein.

[0183] 3. Pharmacogenomics

[0184] Agents, or modulators which have a stimulatory or inhibitoryeffect on MDA-9 activity (e.g., MDA-9 gene expression) as identified bya screening assay described herein can be administered to individuals totreat (prophylactically or therapeutically) disorders (e.g.,drug-resistance) associated with aberrant MDA-9 activity. In conjunctionwith such treatment, the pharmacogenomics (i.e., the study of therelationship between an individual's genotype and that individual'sresponse to a foreign compound or drug) of the individual may beconsidered. Differences in metabolism of therapeutics can lead to severetoxicity or therapeutic failure by altering the relation between doseand blood concentration of the pharmacologically active drug. Thus, thepharmacogenomics of the individual permits the selection of effectiveagents (e.g., drugs) for prophylactic or therapeutic treatments based ona consideration of the individual's genotype. Such pharmacogenomics canfurther be used to determine appropriate dosages and therapeuticregimens. Accordingly, the activity of MDA-9 protein, expression ofMDA-9 nucleic acid, or mutation content of MDA-9 genes in an individualcan be determined to thereby select appropriate agent(s) for therapeuticor prophylactic treatment of the individual.

[0185] Pharmacogenomics deals with clinically significant hereditaryvariations in the response to drugs due to altered drug disposition andabnormal action in affected persons. See, e.g., Linder (1997) Clin.Chem. 43(2):254-266. In general, two types of pharmacogenetic conditionscan be differentiated. Genetic conditions transmitted as a single factoraltering the way drugs act on the body (altered drug action) or geneticconditions transmitted as single factors altering the way the body actson drugs (altered drug metabolism). These pharmacogenetic conditions canoccur either as rare defects or as polymorphisms. For example,glucose-6-phosphate dehydrogenase deficiency (G6PD) is a commoninherited enzymopathy in which the main clinical complication ishaemolysis after ingestion of oxidant drugs (anti-malarials,sulfonamides, analgesics, nitrofurans) and consumption of fava beans.

[0186] As an illustrative embodiment, the activity of drug metabolizingenzymes is a major determinant of both the intensity and duration ofdrug action. The discovery of genetic polymorphisms of drug metabolizingenzymes (e.g., N-acetyltransferase 2 (NAT 2) and cytochrome P450 enzymesCYP2D6 and CYP2C19) has provided an explanation as to why some patientsdo not obtain the expected drug effects or show exaggerated drugresponse and serious toxicity after taking the standard and safe dose ofa drug. These polymorphisms are expressed in two phenotypes in thepopulation, the extensive metabolizer (EM) and poor metabolizer (PM).The prevalence of PM is different among different populations. Forexample, the gene coding for CYP2D6 is highly polymorphic and severalmutations have been identified in PM, which all lead to the absence offunctional CYP2D6. Poor metabolizers of CYP2D6 and CYP2C 19 quitefrequently experience exaggerated drug response and side effects whenthey receive standard doses. If a metabolite is the active therapeuticmoiety, PM show no therapeutic response, as demonstrated for theanalgesic effect of codeine mediated by its CYP2D6-formed metabolitemorphine. The other extreme are the so called ultra-rapid metabolizerswho do not respond to standard doses. Recently, the molecular basis ofultra-rapid metabolism has been identified to be due to CYP2D6 geneamplification.

[0187] Thus, the activity of MDA-9 protein, expression of MDA-9 nucleicacid, or mutation content of MDA-9 genes in an individual can bedetermined to thereby select appropriate agent(s) for therapeutic orprophylactic treatment of the individual. In addition, pharmacogeneticstudies can be used to apply genotyping of polymorphic alleles encodingdrug-metabolizing enzymes to the identification of an individual's drugresponsiveness phenotype. This knowledge, when applied to dosing or drugselection, can avoid adverse reactions or therapeutic failure and thusenhance therapeutic or prophylactic efficiency when treating a subjectwith a MDA-9 modulator, such as a modulator identified by one of theexemplary screening assays described herein.

[0188] 4. Monitoring of Effects During Clinical Trials

[0189] Monitoring the influence of agents (e.g., drugs, compounds) onthe expression or activity of MDA-9 (e.g., the ability to modulate thedrug-resistant phenotype of a cell) can be applied not only in basicdrug screening, but also in clinical trials. For example, theeffectiveness of an agent determined by a screening assay as describedherein to decrease MDA-9 gene expression, protein levels, ordownregulate MDA-9 activity, can be monitored in clinical trails ofsubjects exhibiting increased MDA-9 gene expression, protein levels, orupregulated MDA-9 activity. Alternatively, the effectiveness of an agentdetermined by a screening assay to increase MDA-9 gene expression,protein levels, or upregulate MDA-9 activity (e.g., to increase the drugresistance of a non-cancerous cell), can be monitored in clinical trialsof compounds designed to increase MDA-9 gene expression, protein levels,or upregulate MDA-9 activity. In such clinical trials, the expression oractivity of MDA-9 and, preferably, other genes that have been implicatedin, for example, a cellular proliferation disorder, can be used as a“read out” or markers of the drug resistance of a particular cell.

[0190] For example, and not by way of limitation, genes, includingMDA-9, that are modulated in cells by treatment with an agent (e.g.,compound, drug or small molecule) which modulates MDA-9 activity (e.g.,identified in a screening assay as described herein) can be identified.Thus, to study the effect of agents on cellular proliferation disorders,for example, in a clinical trial, cells can be isolated and RNA preparedand analyzed for the levels of expression of MDA-9 and other genesimplicated in the disorder. The levels of gene expression (i.e., a geneexpression pattern) can be quantified by Northern blot analysis orRT-PCR, as described herein, or alternatively by measuring the amount ofprotein produced, by one of the methods as described herein, or bymeasuring the levels of activity of MDA-9 or other genes. In this way,the gene expression pattern can serve as a marker, indicative of thephysiological response of the cells to the agent. Accordingly, thisresponse state may be determined before, and at various points during,treatment of the individual with the agent.

[0191] In a preferred embodiment, the present invention provides amethod for monitoring the effectiveness of treatment of a subject withan agent (e.g., an agonist, antagonist, peptidomimetic, protein,peptide, nucleic acid, small molecule, or other drug candidateidentified by the screening assays described herein) comprising thesteps of (i) obtaining a pre-administration sample from a subject priorto administration of the agent; (ii) detecting the level of expressionof a MDA-9 protein, mRNA, or genomic DNA in the preadministrationsample; (iii) obtaining one or more post-administration samples from thesubject; (iv) detecting the level of expression or activity of the MDA-9protein, mRNA, or genomic DNA in the post-administration samples; (v)comparing the level of expression or activity of the MDA-9 protein,mRNA, or genomic DNA in the pre-administration sample with the MDA-9protein, mRNA, or genomic DNA in the post administration sample orsamples; and (vi) altering the administration of the agent to thesubject accordingly. For example, increased administration of the agentmay be desirable to decrease the expression or activity of MDA-9 tohigher levels than detected, i.e., to increase the effectiveness of theagent.

[0192] C. Methods of Treatment

[0193] The present invention provides for both prophylactic andtherapeutic methods of treating a subject at risk of (or susceptible to)a disorder or having a disorder associated with aberrant MDA-9expression or activity. Such disorders include cellular resistance tochemotherapeutic drugs.

[0194] 1. Prophylactic Methods

[0195] In one aspect, the invention provides a method for preventing ina subject, a disease or condition associated with an aberrant MDA-9expression or activity (e.g., the development of drug resistance), byadministering to the subject an agent which modulates MDA-9 expressionor at least one MDA-9 activity. Subjects at risk for a condition whichis caused or contributed to by aberrant MDA-9 expression or activity canbe identified by, for example, any or a combination of diagnostic orprognostic assays as described herein. Administration of a prophylacticagent can occur prior to the manifestation of symptoms characteristic ofthe MDA-9 aberrancy, such that a disease or disorder is prevented or,alternatively, delayed in its progression. For example, administrationof a prophylatic agent to a cancer patient may prevent or delay thedevelopment of drug resistance in the patient's cancer cells. Dependingon the type of MDA-9 aberrancy, for example, a MDA-9 agonist or MDA-9antagonist agent can be used for treating the subject. The appropriateagent can be determined based on screening assays described herein.

[0196] 2. Therapeutic Methods

[0197] Another aspect of the invention pertains to methods of modulatingMDA-9 expression or activity for therapeutic purposes. For example, theeffectiveness of chemotherapy is “potentiated” (enhanced) by restoringor improving vulnerability of the transformed cells to the cytotoxiceffects of a chemotherapeutic drug that otherwise would be lesseffective by reducing the expression of MDA-9 in the cells. Themodulatory method of the invention involves contacting a cell with anagent that modulates one or more of the activities of MDA-9 proteinactivity associated with the cell. An agent that modulates MDA-9 proteinactivity can be an agent as described herein, such as a nucleic acid ora protein, a naturally-occurring cognate ligand of a MDA-9 protein, apeptide, a MDA-9 peptidomimetic, or other small molecule. In oneembodiment, the agent stimulates one or more of the biologicalactivities of MDA-9 protein. Examples of such stimulatory agents includeactive MDA-9 protein and a nucleic acid molecule encoding MDA-9 that hasbeen introduced into the cell. In another embodiment, the agent inhibitsone or more of the biological activities of MDA-9 protein. Examples ofsuch inhibitory agents include antisense MDA-9 nucleic acid moleculesand anti-MDA-9 antibodies. These modulatory methods can be performed invitro (e.g., by culturing the cell with the agent) or, alternatively, invivo (e.g, by administering the agent to a subject). As such, thepresent invention provides methods of treating an individual afflictedwith a disease or disorder characterized by aberrant expression oractivity of a MDA-9 protein or nucleic acid molecule. In one embodiment,the method involves administering an agent (e.g., an agent identified bya screening assay described herein), or combination of agents thatmodulates (e.g., upregulates or downregulates) MDA-9 expression oractivity. In another embodiment, the method involves administering aMDA-9 protein or nucleic acid molecule as therapy to compensate forreduced or aberrant MDA-9 expression or activity.

[0198] For example, in one embodiment, the method involves administeringthe desired drug (e.g., cyclophosphamide) to an individual afflictedwith a drug-resistant cell population (a tumor, e.g., a carcinoma,sarcoma, leukemia, lymphoma, or lymphosarcoma), and coadministering aninhibitor of MDA-9 expression or activity. The administration andcoadministration steps can be carried out concurrently or in any order,and can be separated by a time interval sufficient to allow uptake ofeither compound by the cells to be eradicated. For example, an antisensepharmaceutical composition (or a cocktail composition comprising anMDA-9 antisense oligonucleotide in combination with one or more otherantisense oligonucleotides) can be administered to the individualsufficiently in advance of administration of the chemotherapeutic drugto allow the antisense composition to permeate the individual's tissues,especially tissue comprising the transformed cells to be eradicated; tobe internalized by transformed cells; and to disrupt MDA-9 geneexpression and/or protein production.

[0199] Inhibition of MDA-9 activity is desirable in situations in whichMDA-9 is abnormally upregulated and/or in which decreased MDA-9 activityis likely to have a beneficial effect. Conversely, stimulation of MDA-9activity is desirable in situations in which MDA-9 is abnormallydownregulated and/or in which increased MDA-9 activity is likely to havea beneficial effect.

[0200] This invention is further illustrated by the following exampleswhich should not be construed as limiting. The contents of allreferences, patents and published patent applications cited throughoutthis application are hereby incorporated by reference.

EXAMPLES Example 1: Isolation and Characterization of a Human MDA-9 CDNA

[0201] In order to identify genes that are more highly expressed in drugresistant cells than non-drug resistant cells, the expression of anumber of genes was measured in drug-resistant cell lines and therelated drug-sensitive cell lines. This led to the identification ofclone cohyr002g08. Northern analysis revealed that this clone isexpressed at a higher level in three doxorubicin resistant cancer celllines, HL-60 ADR, A2780 ADR, and U937 A200, than in the correspondingdrug-sensitive cell lines from which the drug-resistant cell lines werederived.

[0202] A sequence homology serach led to the identification of thisclone as melanoma differentiation associated gene 9 (Genbank AccessionNo. AF006636; Lin et al. (1996) Molecular and Cellular Differentiation4:317-333). The nucleic acid and amino acid sequences of MDA-9 are shownin FIGS. 1 and 2 respectively.

[0203] The amino aicd sequence of MDA-9 is identical to the amino aicdsequence of syntenin (GenBank Accession Number AF000652; David et al.(1997) Proc. Nat'l. Acad. Sci USA 94:13683-88). Thus, MDA-9, likesyntenin, may act as a scaffolding protein. If so, MDA-9 could be anadaptor molecule linking the plasma membrane with signalling molecules.Thus, MDA-9 might act as an organizer of transmembrane proteins (e.g.,MDR1/MRPs) or as a recruiter of proteins from the cytosol. MDA-9 alsohas some sequence similarity to Pbp1 (GenBank Accession No. U83463),also a scaffolding protein.

Example 2: Preparation of MDA-9 Proteins

[0204] Recombinant MDA-9 can be produced in a variety of expressionsystems. For example, the mature MDA-9 peptide can be expressed as arecombinant glutathione-S-transferase (GST) fusion protein in E. coliand the fusion protein can be isolated and characterized. Specifically,as described above, MDA-9 can be fused to GST and this fusion proteincan be expressed in E. coli strain PEB199. As MDA-9 is predicted to be44,960 Da and GST is predicted to be 26,000 Da, the fusion protein ispredicted to be 70,960 Da in molecular weight. Expression of theGST-MDA-9 fusion protein in PEB199 can be induced with IPTG. Therecombinant fusion protein can be purified from crude bacterial lysatesof the induced PEB199 strain by affinity chromatography on glutathionebeads. Using polyacrylamide gel electrophoretic analysis of the proteinspurified from the bacterial lysates, the resultant fusion protein shouldbe 70,960 Da in size.

[0205] Equivalents

[0206] Those skilled in the art will recognize, or be able to ascertainusing no more than routine experimentation, many equivalents to thespecific embodiments of the invention described herein. Such equivalentsare intended to be encompassed by the following claims.

1 3 1 2068 DNA Homo sapiens CDS (76)...(970) 1 cctcagaagt ccgtgccagtgaccggaggc ggcggcggcg agcggttcct tgtgggctag 60 aagaatcctg caaaa atg tctctc tat cca tct ctc gaa gac ttg aag gta 111 Met Ser Leu Tyr Pro Ser LeuGlu Asp Leu Lys Val 1 5 10 gac aaa gta att cag gct caa act gct ttt tctgca aac cct gcc aat 159 Asp Lys Val Ile Gln Ala Gln Thr Ala Phe Ser AlaAsn Pro Ala Asn 15 20 25 cca gca att ttg tca gaa gct tct gct cct atc cctcac gat gga aat 207 Pro Ala Ile Leu Ser Glu Ala Ser Ala Pro Ile Pro HisAsp Gly Asn 30 35 40 ctc tat ccc aga ctg tat cca gag ctc tct caa tac atgggg ctg agt 255 Leu Tyr Pro Arg Leu Tyr Pro Glu Leu Ser Gln Tyr Met GlyLeu Ser 45 50 55 60 tta aat gaa gaa gaa ata cgt gca aat gtg gcc gtg gtttct ggt gca 303 Leu Asn Glu Glu Glu Ile Arg Ala Asn Val Ala Val Val SerGly Ala 65 70 75 cca ctt cag ggg cag ttg gta gca aga cct tcc agt ata aactat atg 351 Pro Leu Gln Gly Gln Leu Val Ala Arg Pro Ser Ser Ile Asn TyrMet 80 85 90 gtg gct cct gta act ggt aat gat gtt gga att cgt aga gca gaaatt 399 Val Ala Pro Val Thr Gly Asn Asp Val Gly Ile Arg Arg Ala Glu Ile95 100 105 aag caa ggg att cgt gaa gtc att ttg tgt aag gat caa gat ggaaaa 447 Lys Gln Gly Ile Arg Glu Val Ile Leu Cys Lys Asp Gln Asp Gly Lys110 115 120 att gga ctc agg ctt aaa tca ata gat aat ggt ata ttt gtt cagcta 495 Ile Gly Leu Arg Leu Lys Ser Ile Asp Asn Gly Ile Phe Val Gln Leu125 130 135 140 gtc cag gct aat tct cca gcc tca ttg gtt ggt ctg aga tttggg gac 543 Val Gln Ala Asn Ser Pro Ala Ser Leu Val Gly Leu Arg Phe GlyAsp 145 150 155 caa gta ctt cag atc aat ggt gaa aac tgt gca gga tgg agctct gat 591 Gln Val Leu Gln Ile Asn Gly Glu Asn Cys Ala Gly Trp Ser SerAsp 160 165 170 aaa gcg cac aag gtg ctc aaa cag gct ttt gga gag aag attacc atg 639 Lys Ala His Lys Val Leu Lys Gln Ala Phe Gly Glu Lys Ile ThrMet 175 180 185 acc att cgt gac agg ccc ttt gaa cgg acg att acc atg cataag gat 687 Thr Ile Arg Asp Arg Pro Phe Glu Arg Thr Ile Thr Met His LysAsp 190 195 200 agc act gga cat gtt ggt ttt atc ttt aaa aat gga aaa ataaca tcc 735 Ser Thr Gly His Val Gly Phe Ile Phe Lys Asn Gly Lys Ile ThrSer 205 210 215 220 ata gtg aaa gat agc tct gca gcc aga aat ggt ctt ctcacg gaa cat 783 Ile Val Lys Asp Ser Ser Ala Ala Arg Asn Gly Leu Leu ThrGlu His 225 230 235 aac atc tgt gaa atc aat gga cag aat gtc att gga ttgaag gac tct 831 Asn Ile Cys Glu Ile Asn Gly Gln Asn Val Ile Gly Leu LysAsp Ser 240 245 250 caa att gca gac ata ctg tca aca tct ggg act gta gttact att aca 879 Gln Ile Ala Asp Ile Leu Ser Thr Ser Gly Thr Val Val ThrIle Thr 255 260 265 atc atg cct gct ttt atc ttt gaa cat att att aag cggatg gca cca 927 Ile Met Pro Ala Phe Ile Phe Glu His Ile Ile Lys Arg MetAla Pro 270 275 280 agc att atg aaa agc cta atg gac cac acc att cct gaggtt t 970 Ser Ile Met Lys Ser Leu Met Asp His Thr Ile Pro Glu Val 285290 295 aaaattcacg gcaccatgga aatgtagctg aacgtctcca gtttccttctttggcaactt 1030 ctgtattatg cacgtgaagc cttcccggag ccagcgagca tatgctgcatgaggaccttt 1090 ctatcttaca ttatggctgg gaatcttact ctttcatctg ataccttgttcagatttcaa 1150 aatagttgta gccttatcct ggttttacag atgtgaaact ttcaagagatttactgactt 1210 tcctagaata gtttctctac tggaaacctg atgcttttat aagccattgtgattaggatg 1270 actgttacag gcttagcttt gtgtgaaaac cagtcacctt tctcctaggtaatgagtagt 1330 gctgttcata ttactttagt tctatagcat actgcatctt taacatgctatcatagtaca 1390 tttagaatga ttgcctttga tttttttttt aaattctgtg tgtgtgtgtgtaaaatgcca 1450 attaagaaca ctggtttcat tccatgtaag cattaaacag tgtatgtaggtttcaagaga 1510 ttgtgatgat tcttaaattt taactacctt cacttaatat gcttgaactgtcgccttaac 1570 tatgttaagc atctagacta aaagccaaaa tataattatt gctgcctttctaaaaaccca 1630 aaatgtagtt ctctattaac ctgaaatgta cactagccca gaacagtttaatggtactta 1690 ctgagctata gcatagctgc ttagttgttt ttgagagttt ttagtcaacacataatggaa 1750 acttctttct tctaaaagtt gccagtgcca cttttaagaa gtgaatcactatatgtgatg 1810 taaaagttat tacactaaac aggataaact tttgactccc cttttgttcatttgtggatt 1870 aagtggtata atacttaatt ttggcatttg actcttaaga ttatgtaacctagctacttt 1930 gggatggtct tagaatattt ttctgataac ttgttccttt tcctgactcctccttgcaaa 1990 caaaatgata gttgacactt tatcctgatt tttttcttct ttttggtttatgtctattct 2050 aattaaatat gtataaat 2068 2 298 PRT Homo sapiens 2 MetSer Leu Tyr Pro Ser Leu Glu Asp Leu Lys Val Asp Lys Val Ile 1 5 10 15Gln Ala Gln Thr Ala Phe Ser Ala Asn Pro Ala Asn Pro Ala Ile Leu 20 25 30Ser Glu Ala Ser Ala Pro Ile Pro His Asp Gly Asn Leu Tyr Pro Arg 35 40 45Leu Tyr Pro Glu Leu Ser Gln Tyr Met Gly Leu Ser Leu Asn Glu Glu 50 55 60Glu Ile Arg Ala Asn Val Ala Val Val Ser Gly Ala Pro Leu Gln Gly 65 70 7580 Gln Leu Val Ala Arg Pro Ser Ser Ile Asn Tyr Met Val Ala Pro Val 85 9095 Thr Gly Asn Asp Val Gly Ile Arg Arg Ala Glu Ile Lys Gln Gly Ile 100105 110 Arg Glu Val Ile Leu Cys Lys Asp Gln Asp Gly Lys Ile Gly Leu Arg115 120 125 Leu Lys Ser Ile Asp Asn Gly Ile Phe Val Gln Leu Val Gln AlaAsn 130 135 140 Ser Pro Ala Ser Leu Val Gly Leu Arg Phe Gly Asp Gln ValLeu Gln 145 150 155 160 Ile Asn Gly Glu Asn Cys Ala Gly Trp Ser Ser AspLys Ala His Lys 165 170 175 Val Leu Lys Gln Ala Phe Gly Glu Lys Ile ThrMet Thr Ile Arg Asp 180 185 190 Arg Pro Phe Glu Arg Thr Ile Thr Met HisLys Asp Ser Thr Gly His 195 200 205 Val Gly Phe Ile Phe Lys Asn Gly LysIle Thr Ser Ile Val Lys Asp 210 215 220 Ser Ser Ala Ala Arg Asn Gly LeuLeu Thr Glu His Asn Ile Cys Glu 225 230 235 240 Ile Asn Gly Gln Asn ValIle Gly Leu Lys Asp Ser Gln Ile Ala Asp 245 250 255 Ile Leu Ser Thr SerGly Thr Val Val Thr Ile Thr Ile Met Pro Ala 260 265 270 Phe Ile Phe GluHis Ile Ile Lys Arg Met Ala Pro Ser Ile Met Lys 275 280 285 Ser Leu MetAsp His Thr Ile Pro Glu Val 290 295 3 894 DNA Homo sapiens 3 atgtctctctatccatctct cgaagacttg aaggtagaca aagtaattca ggctcaaact 60 gctttttctgcaaaccctgc caatccagca attttgtcag aagcttctgc tcctatccct 120 cacgatggaaatctctatcc cagactgtat ccagagctct ctcaatacat ggggctgagt 180 ttaaatgaagaagaaatacg tgcaaatgtg gccgtggttt ctggtgcacc acttcagggg 240 cagttggtagcaagaccttc cagtataaac tatatggtgg ctcctgtaac tggtaatgat 300 gttggaattcgtagagcaga aattaagcaa gggattcgtg aagtcatttt gtgtaaggat 360 caagatggaaaaattggact caggcttaaa tcaatagata atggtatatt tgttcagcta 420 gtccaggctaattctccagc ctcattggtt ggtctgagat ttggggacca agtacttcag 480 atcaatggtgaaaactgtgc aggatggagc tctgataaag cgcacaaggt gctcaaacag 540 gcttttggagagaagattac catgaccatt cgtgacaggc cctttgaacg gacgattacc 600 atgcataaggatagcactgg acatgttggt tttatcttta aaaatggaaa aataacatcc 660 atagtgaaagatagctctgc agccagaaat ggtcttctca cggaacataa catctgtgaa 720 atcaatggacagaatgtcat tggattgaag gactctcaaa ttgcagacat actgtcaaca 780 tctgggactgtagttactat tacaatcatg cctgctttta tctttgaaca tattattaag 840 cggatggcaccaagcattat gaaaagccta atggaccaca ccattcctga ggtt 894

What is claimed is:
 1. A method for determining whether a test compoundmodulates the drug resistance of a cell, the method comprising: a)determining the level of MDA-9 expression in a cell in the presence of atest compound; b) determining the level of MDA-9 expression in the cellin the absence of the test compound; and c) identifying the compound asa modulator of drug resistance of the cell if the level of expression ofMDA-9 in the cell in the presence of the test compound differs from thelevel of expression of MDA-9 in the cell in the absence of the testcompound.
 2. The method of claim 1 wherein the MDA-9 is encoded by anendogenous gene.
 3. A method for determining whether a test compoundmodulates the drug resistance of a cell, the method comprising: a)incubating MDA-9 protein in the presence of a test compound; b)determining whether the test compound binds to the MDA-9 protein; c)selecting a test compound which binds to the MDA-9 protein; d)administering the test compound selected in step c) to a non-humanmammal having drug resistant cells; e) determining whether the testcompound alters the drug resistance of the cells in the non-humanmammal; and f) identifying the test compound as a modulator of drugresistance of the cell if the compound alters the drug resistance of thecells in step (e).
 4. A method for determining whether a test cell has adrug-resistant phenotype, the method comprising: a) measuring theexpression of MDA-9 in the test cell; b) comparing the expression ofMDA-9 measured in step a) to the expression of MDA-9 in a control cellnot having a drug-resistant phenotype; and c) determining that the testcell has a drug resistant phenotype if the expression of MDA-9 in thetest cell is greater than the expression of MDA-9 in the control cell.5. A method of determining whether a test cell has a drug-resistantphenotype, the method comprising: a) measuring the activity of MDA-9 inthe test cell; b) comparing the activity of MDA-9 measured in step a) tothe activity of MDA-9 in a control cell not having a drug-resistantphenotype; and c) determining that the test cell has a drug resistantphenotype if the activity of MDA-9 in the test cell is greater than theactivity of MDA-9 in the control cell.
 6. A method for determiningwhether a subject has or is at risk of developing a drug resistanttumor, the method comprising: a) measuring the expression of MDA-9 mRNAin a biological sample obtained from the subject; b) comparing theexpression of MDA-9 mRNA measured in step a) to the expression of MDA-9mRNA in a biological sample obtained from a control subject not having adrug resistant tumor; and c) determining that the patient has or is atrisk of developing a drug resistant tumor if the expression of MDA-9mRNA in the biological sample obtained from the patient is higher thanthe expression of MDA-9 mRNA in the biological sample obtained from thecontrol subject.
 7. The method of claim 6, wherein step a) comprises theuse of a nucleic acid molecule that hybridizes to MDA-9 mRNA.
 8. Amethod for determining whether a subject has or is at risk of developinga drug resistant tumor, the method comprising: a) measuring the activityof MDA-9 in a biological sample obtained from the subject; b) comparingthe activity of MDA-9 measured in step a) to the expression of MDA-9mRNA in a biological sample obtained from a control subject not having adrug resistant tumor; and c) determining that the patient has or is atrisk of developing a drug resistant tumor if the activity of MDA-9 inthe biological sample obtained from the patient is higher than theactivity of MDA-9 in the biological sample obtained from the controlsubject.
 9. The method of claim 8, wherein step a) comprises the use ofan agent that binds to MDA-9 protein.
 10. A method for monitoring theeffect of an anti-tumor treatment on a patient, the method comprising:a) measuring the expression of MDA-9 in a tumor sample obtained from thepatient; b) comparing the expression of MDA-9 measured in step a) to theexpression of MDA-9 in a control sample of cells; and c) determiningthat the anti-tumor treatment should be discontinued or modified if theexpression of MDA-9 in the tumor sample is higher than the expression ofMDA-9 in the control sample of cells.
 11. The method of claim 10,wherein step a) comprises the use of a nucleic acid molecule thathybridizes to MDA-9 mRNA.
 12. A method for monitoring the effect of ananti-tumor treatment on a patient, the method comprising: a) measuringthe activity of MDA-9 in a tumor sample obtained from the patient; b)comparing the activity of MDA-9 measured in step a) to the activity ofMDA-9 in a control sample of cells; and c) determining that theanti-tumor treatment should be discontinued or modified if the activityof MDA-9 in the tumor sample is higher than the activity of MDA-9 in thecontrol sample of cells.
 13. The method of claim 12, wherein step a)comprises the use of an agent that binds to MDA-9 protein.
 14. A methodfor modulating the drug resistance of a cell, the method comprisingmodulating MDA-9 expression within the cell.
 15. A method reducing thedrug resistance of cell, the method comprising contacting the cell witha molecule which reduces the expression of MDA-9 within the cell.
 16. Amethod of increasing the effectiveness of a chemotherapeutic compound ina patient suffering from a disorder associated with the presence ofdrug-resistant neoplastic cells, the method comprising: a) administeringa chemotherapeutic compound to the patient; and b) administering acompound with reduces MDA-9 expression to the patient.
 17. A method oftreating a mammal suspected of having a disorder associated with thepresence of drug-resistant cells, the method comprising administering tothe mammal a compound that reduces the expression of MDA-9 in thedrug-resistant cells, the reduction be sufficient to reduce the drugresistance of the drug resistant cells.
 18. A method for increasing thedrug resistance of cell that has an undesirably low level of MDA-9expression, the method comprising exposing the cell to a compound thatincreases the expression of MDA-9.
 19. A method for treating a drugresistant tumor in a patient, the method comprising administering tosaid subject an amount of a MDA-9 antagonist effective to reduce drugresistance of said tumor in the patient.
 20. The use of an inhibitor ofMDA-9 expression, or pharmaceutically acceptable salt thereof, or apharmaceutical composition containing either entity, for the manufactureof a medicament for the treatment of a drug resistant tumor in apatient.